Compositions comprising a lipolytic enzyme variant and methods of use thereof

ABSTRACT

The present disclosure provides lipolytic enzyme variants having two or more modifications as compared to a parent lipolytic enzyme. Specifically, the present disclosure provides one or more lipolytic enzyme variants that has at least one improved performance when compared to one or more reference lipolytic enzymes. In addition, the present disclosure provides compositions comprising a lipolytic enzyme variant of the disclosure. The present disclosure also provides methods of cleaning using compositions comprising a lipolytic enzyme variant of the disclosure.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/960,353, filed Jan. 13, 2020, incorporated herein in its entirety byreference.

FIELD

The disclosure relates to lipolytic enzyme variants, polynucleotidesencoding the variants, methods of producing the variants, and methods ofusing the variants.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named20200109_NB41780USPSP_SeqLst.txt created on Jan. 9, 2020 and having asize 7 kilobytes and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

BACKGROUND

Lipolytic enzymes have been employed in detergent cleaning compositionsfor the removal of oily stains. One mechanism by which lipolytic enzymesfunction is by hydrolyzing triglycerides to generate fatty acids.However, these enzymes are often inhibited by surfactants and othercomponents present in cleaning composition, interfering with theirability to remove oily stains. Accordingly, the need exists forlipolytic enzymes that can function in the harsh environment of cleaningcompositions.

SUMMARY

The present disclosure provides one or more lipolytic enzyme variants,and compositions and methods related to the production and use thereof,including one or more lipolytic enzyme variants that has at least oneimproved performance when compared to one or more reference lipolyticenzymes. Specifically, the present disclosure provides lipolytic enzymevariants having two or more modifications, such as a substitution, ascompared to a parent lipolytic enzyme. The improved performance of theone or more lipolytic enzyme variants or active fragments thereofrelative to the parent lipolytic enzyme, can be an improved washperformance, a decreased malodor, an increased detergent stability, anincreased thermostability, an increased calcium ion binding stability,an increased protease stability, or any one combination thereof. Thepresent disclosure provides lipolytic enzyme variants that areparticularly well suited to and useful in a variety of cleaningapplications. The disclosure also provides methods of cleaning usinglipolytic enzyme variants of the present disclosure.

In one embodiment, the disclosure provides a lipolytic enzyme variant oran active fragment thereof comprising at least two amino acidmodifications to a parent lipolytic enzyme, wherein a first amino acidmodification is at a position of the lipolytic enzyme variant selectedfrom the group consisting of 4, 11, 19, 22, 26, 39, 53, 60, 64, 67, 70,71, 93, 95, 111, 117, 119, 120, 121, 125, 127, 131, 134, 141, 142, 143,146, 149, 154, 159, 163, 166, 170, 179, 185, 196, 199, 203, 204, 205,206, 208, 209, 212, 217, 218, 221, 224, 227, 229, 235, 247, 252, 261,262, 265, 272, 280 and 286, wherein the amino acid positions of thevariant are numbered by correspondence with the amino acid sequence ofSEQ ID NO: 1, wherein the lipolytic enzyme variant has at least 60%sequence identity to the amino acid sequence SEQ ID NO: 1.

In one embodiment, the disclosure provides a lipolytic enzyme variant oractive fragment thereof, wherein the at least two amino acidmodifications to a parent lipolytic enzyme are at a position of thelipolytic enzyme variant selected from the group consisting of 4, 11,19, 22, 26, 39, 53, 60, 64, 67, 70, 71, 93, 95, 111, 117, 119, 120, 121,125, 127, 131, 134, 141, 142, 143, 146, 149, 154, 159, 163, 166, 170,179, 185, 196, 199, 203, 204, 205, 206, 208, 209, 212, 217, 218, 221,224, 227, 229, 235, 247, 252, 261, 262, 265, 272, 280, and 286, whereinthe amino acid positions of the variant are numbered by correspondencewith the amino acid sequence of SEQ ID NO: 1. In one embodiment, thedisclosure provides a lipolytic enzyme variant or active fragmentthereof consisting of two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen or fourteen amino acid modifications to aparent lipolytic enzyme, wherein the two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen or fourteen acidmodifications is at a position of the lipolytic enzyme variant selectedfrom the group consisting of 4, 11, 19, 22, 26, 39, 53, 60, 64, 67, 70,71, 93, 95, 111, 117, 119, 120, 121, 125, 127, 131, 134, 141, 142, 143,146, 149, 154, 159, 163, 166, 170, 179, 185, 196, 199, 203, 204, 205,206, 208, 209, 212, 217, 218, 221, 224, 227, 229, 235, 247, 252, 261,262, 265, 272, 280 and 286, wherein the amino acid positions of thevariant are numbered by correspondence with the amino acid sequence ofSEQ ID NO: 1.

In one aspect, the lipolytic enzyme variant is derived from a parentlipolytic enzyme having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% aminoacid sequence identity to the amino acid sequence of SEQ ID NO: 1. Inone aspect, the variant comprises an amino acid sequence having 60%,65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acidsequence of SEQ ID NO: 1.

In one embodiment, the disclosure provides a lipolytic enzyme variant oran active fragment thereof comprising at least two amino acidmodifications to a parent lipolytic enzyme, wherein a first amino acidmodification is selected from the group consisting of a K at position 4;an R at position 11; an L at position 19; an F at position 22; a C atposition 26; an I at position 39; a Q at position 53; an L at position60; a V or T at position 64; a G at position 67; a V at position 70; anE at position 71; an S at position 93; a Q at position 95; a T atposition 111; a D, E, G, Q or S at position 117; a V at position 119; anE or Q or S or V at position 120; an E or S at position 121; an E atposition 125; a D or E at position 127; a D, E, K or T at position 131;a E or M at position 134; an N at position 141; a V at position 142; anL at position 143; a H at position 146; an I at position 149; a V atposition 154; a D at position 159; a G at position 163; a T at position166; an E or S at position 170; a K at position 179; a Q at position185; a L at position 196; an H at position 199; a C, K, N, R, S ort T atposition 203; a V or Y at position 204; a W at position 205; a C, E or Qat position 206; an E or Q at position 208; an A or D at position 209; aR at position 212; a Y at position 217; a D at position 218; a T atposition 221; an S at position 224; a D or V at position 227; an A, C,H, P, Q, S, T or V at position 229; an A at position 235; an R atposition 247; a D, G, Q or V at position 252; an A or L at positon 261;a C or M at position 262; an A at position 265; a K, P or V at position272; a K at position 280; and a Q at position 286, wherein the aminoacid positions of the variant are numbered by correspondence with theamino acid sequence of SEQ ID NO: 1.

In one embodiment, the disclosure provides a lipolytic enzyme variant oractive fragment thereof comprising at least two amino acid modificationto a parent lipolytic enzyme, wherein said lipolytic enzyme variant oractive fragment thereof has an improved performance relative to theparent lipolytic enzyme, wherein the improved performance is selectedfrom the group consisting of an improved wash performance, a decreasedmalodor, an increased detergent stability, an increased thermostability,an increased calcium ion binding stability, an increased proteasestability, or any one combination thereof. The lipolytic enzyme variantor active fragment thereof having an improved wash performance can be alipolytic enzyme variant that has a wash performance index (PI(wash))relative to the parent lipolytic enzyme that is greater than 1.0. Thelipolytic enzyme variant or active fragment thereof having an increaseddetergent stability can be a lipolytic enzyme variant or active fragmentthereof that has a detergent stability that is greater that thedetergent stability of the parent lipolytic enzyme (scuh as the parentlipolytic enzyme set forth in SEQ ID NO:1).

Some further embodiments are directed to a composition comprising one ormore lipolytic enzyme variants described herein. Further embodiments aredirected to a method of cleaning comprising contacting a surface or anitem in need of cleaning with one or more lipolytic enzyme variantsdescribed herein or one or more compositions described herein.

Still other embodiments are directed to a method for producing a variantdescribed herein, comprising stably transforming a host cell with anexpression vector comprising a polynucleotide encoding one or morelipolytic enzyme variants described herein. Still further embodimentsare directed to a polynucleotide comprising a nucleic acid sequenceencoding one or more lipolytic enzyme variants described herein.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The disclosure can be more fully understood from the following detaileddescription and the accompanying Sequence Listing, which forms a part ofthis application. The sequence descriptions and sequence listingattached hereto comply with the rules governing nucleotide and aminoacid sequence disclosures in patent applications as set forth in 37C.F.R. §§ 1.821 1.825. The sequence descriptions contain the threeletter codes for amino acids as defined in 37 C.F.R. §§ 1.821 1.825,which are incorporated herein by reference.

DETAILED DESCRIPTION

The present disclosure provides one or more lipolytic enzyme variants,and compositions and methods related to the production and use thereof,including one or more lipolytic enzyme variants that has at least oneimproved performance (improved property) when compared to one or morereference lipolytic enzymes. Specifically, the present disclosureprovides lipolytic enzyme variants having two or more modifications,such as a substitution, as compared to a parent lipolytic enzyme. Theimproved performance of one or more lipolytic enzyme variants or activefragments thereof relative to the parent lipolytic enzyme, can be animproved wash performance, a decreased malodor, an increased detergentstability, an increased thermostability, an increased calcium ionbinding stability, an increased protease stability, or any onecombination thereof. The present disclosure provides lipolytic enzymevariants, including, but not limited to, variant lipase lipolyticenzymes, that are particularly well suited to and useful in a variety ofcleaning applications. The disclosure includes compositions comprisingat least one of the lipolytic enzyme variants (e.g., variant lipases)set forth herein. Some such compositions comprise detergentcompositions. The lipolytic enzyme variants of the present disclosurecan be combined with other enzymes useful in detergent compositions.

The disclosure also provides enzyme compositions having a comparable orimproved performance, as compared to known lipolytic enzymes, such as,known lipase lipolytic enzymes (WO2014/059360, WO2015/010009,WO2018/015295). The disclosure also provides methods of cleaning usinglipolytic enzyme variants of the present disclosure.

The disclosure includes enzyme variants of lipolytic enzymes having one,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen or fourteen modifications from a parent lipolytic enzyme. Theenzyme variants can be useful in a composition, such as but not limitingto a detergent composition, by having an improved performance relativeto a parent lipolytic enzyme, wherein the improved performance isselected from the group consisting of an improved wash performance, adecreased malodor, an increased detergent stability, an increasedthermostability, an increased calcium ion binding stability, anincreased protease stability, or any one combination thereof.

The disclosure includes enzyme variants of lipolytic enzymes having twoor more modifications from a parent lipolytic enzyme. These amino acidmodifications can result in an improved detergent stability and/or animproved performance of the variant and their amino acid positions canbe considered useful positions for combinatorial modifications to aparent lipolytic enzyme. Lipolytic enzyme amino acid positions found tobe useful positions can be further characterized by having multiplemodifications that are suitable for use in a composition, such as butnot limiting to a detergent composition.

Unless otherwise indicated herein, one or more lipolytic enzyme variantsdescribed herein can be made and used via conventional techniquescommonly used in molecular biology, microbiology, protein purification,protein engineering, protein and DNA sequencing, recombinant DNA fields,and industrial enzyme use and development.

Terms and abbreviations not defined should be accorded their ordinarymeaning as used in the art. Unless defined otherwise herein, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art. Although manymethods and materials similar or equivalent to those described hereinfind use in the practice of the present disclosure, some methods andmaterials are described herein. All patents, patent applications,articles and publications mentioned herein, both supra and infra, arehereby expressly incorporated herein by reference.

As used herein, the singular terms “a,” “an,” and “the” include theplural reference unless the context clearly indicates otherwise. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively. It is to be understood that thisdisclosure is not limited to the particular methodology, protocols, andreagents described, as these may vary, depending upon the context inwhich they are used by those of skill in the art.

It is intended that every maximum numerical limitation given throughoutthis specification include every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

The terms “derived from” and “obtained from” refer not only to alipolytic enzyme produced or producible by a strain of the organism inquestion, but also a lipolytic enzyme encoded by a DNA sequence isolatedfrom such strain and produced in a host organism containing such DNAsequence. Additionally, the term refers to a lipolytic enzyme which isencoded by a DNA sequence of synthetic and/or cDNA origin and which hasthe identifying characteristics of the lipolytic enzyme in question. Toexemplify, “lipolytic enzymes derived from Proteus” refers to thoseenzymes having lipolytic activity which are naturally produced byProteus, as well as to lipolytic enzymes like those produced by Proteussources but which through the use of genetic engineering techniques areproduced by non-Proteus organisms transformed with a nucleic acidencoding the lipolytic enzymes.

The term “vector” refers to a nucleic acid construct used to introduceor transfer nucleic acid(s) into a target cell or tissue. A vector istypically used to introduce foreign DNA into a cell or tissue. Vectorsinclude plasmids, cloning vectors, bacteriophages, viruses (e.g., viralvector), cosmids, expression vectors, shuttle vectors, and the like. Avector typically includes an origin of replication, a multicloning site,and a selectable marker. The process of inserting a vector into a targetcell is typically referred to as transformation.

As used herein in the context of introducing a nucleic acid sequenceinto a cell, the term “introduced” refers to any method suitable fortransferring the nucleic acid sequence into the cell. Such methods forintroduction include but are not limited to protoplast fusion,transfection, transformation, electroporation, conjugation, andtransduction. Transformation refers to the genetic alteration of a cellwhich results from the uptake, optional genomic incorporation, andexpression of genetic material (e.g., DNA).

The term “expression” refers to the transcription and stableaccumulation of sense (mRNA) or anti-sense RNA, derived from a nucleicacid molecule of the disclosure. Expression may also refer totranslation of mRNA into a polypeptide. Thus, the term “expression”includes any step involved in the “production of the polypeptide”including, but not limited to, transcription, post-transcriptionalmodifications, translation, post-translational modifications, secretionand the like.

The phrases “expression cassette” or “expression vector” refers to anucleic acid construct or vector generated recombinantly orsynthetically for the expression of a nucleic acid of interest (e.g., aforeign nucleic acid or transgene) in a target cell. The nucleic acid ofinterest typically expresses a protein of interest. An expression vectoror expression cassette typically comprises a promoter nucleotidesequence that drives or promotes expression of the foreign nucleic acid.The expression vector or cassette also typically includes otherspecified nucleic acid elements that permit transcription of aparticular nucleic acid in a target cell. A recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Some expressionvectors have the ability to incorporate and express heterologous DNAfragments in a host cell or genome of the host cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors for expression of a protein from anucleic acid sequence incorporated into the expression vector is withinthe knowledge of those of skill in the art.

As used herein, a nucleic acid is “operably linked” with another nucleicacid sequence when it is placed into a functional relationship withanother nucleic acid sequence. For example, a promoter or enhancer isoperably linked to a nucleotide coding sequence if the promoter affectsthe transcription of the coding sequence. A ribosome binding site may beoperably linked to a coding sequence if it is positioned so as tofacilitate translation of the coding sequence. Typically, “operablylinked” DNA sequences are contiguous. However, enhancers do not have tobe contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, synthetic oligonucleotideadaptors or linkers may be used in accordance with conventionalpractice.

The term “gene” refers to a polynucleotide (e.g., a DNA segment), thatencodes a polypeptide and includes regions preceding and following thecoding regions. In some instances, a gene includes intervening sequences(introns) between individual coding segments (exons).

The terms “host strain” and “host cell” refer to a suitable host for anexpression vector comprising a DNA sequence of interest.

The term “recombinant”, when used with reference to a cell typicallyindicates that the cell has been modified by the introduction of aforeign nucleic acid sequence or that the cell is derived from a cell somodified. For example, a recombinant cell may comprise a gene not foundin identical form within the native (non-recombinant) form of the cell,or a recombinant cell may comprise a native gene (found in the nativeform of the cell) that has been modified and re-introduced into thecell. A recombinant cell may comprise a nucleic acid endogenous to thecell that has been modified without removing the nucleic acid from thecell; such modifications include those obtained by gene replacement,site-specific mutation, and related techniques known to those ofordinary skill in the art. Recombinant DNA technology includestechniques for the production of recombinant DNA in vitro and transferof the recombinant DNA into cells where it may be expressed orpropagated, thereby producing a recombinant polypeptide. “Recombination”and “recombining” of polynucleotides or nucleic acids refer generally tothe assembly or combining of two or more nucleic acid or polynucleotidestrands or fragments to generate a new polynucleotide or nucleic acid.

A nucleic acid or polynucleotide is said to “encode” a polypeptide if,in its native state or when manipulated by methods known to those ofskill in the art, it can be transcribed and/or translated to produce thepolypeptide or a fragment thereof. The anti-sense strand of such anucleic acid is also said to encode the sequence.

A “protein” or “polypeptide” comprises a polymeric sequence of aminoacid residues. The terms “protein” and “polypeptide” are usedinterchangeably herein. The single and 3-letter code for amino acids asdefined in conformity with the IUPAC-IUB Joint Commission on BiochemicalNomenclature (JCBN) is used through out this disclosure. It is alsounderstood that a polypeptide can be coded for by more than onenucleotide sequence due to the degeneracy of the genetic code. Mutationscan be named by the one letter code for the parent amino acid, followedby a number and then the one letter code for the variant amino acid. Forexample, mutating glycine (G) at position 87 to serine (S) can berepresented as “G087S” or “G87S”. Multiple mutations can be indicated byinserting a “_” between the mutations. For example, mutations atpositions 87 and 90 can be represented as either “G087S_A090Y” or“G87S_A90Y” or “G87S+A90Y” or “G087S+A090Y”.

The terms “signal sequence” and “signal peptide” refer to a sequence ofamino acid residues that may participate in the secretion or directtransport of the mature or precursor form of a protein. The signalsequence is typically located N-terminal to the precursor or matureprotein sequence. The signal sequence may be endogenous or exogenous. Asignal sequence is normally absent from the mature protein. A signalsequence is typically cleaved from the protein by a signal peptidaseafter the protein is transported.

The term “mature” form of a protein, polypeptide, or peptide refers tothe functional form of the protein, polypeptide, or peptide without thesignal peptide sequence and propeptide sequence.

The term “precursor” form of a protein or peptide refers to a matureform of the protein having a prosequence operably linked to the amino orcarbonyl terminus of the protein. The precursor may also have a “signal”sequence operably linked to the amino terminus of the prosequence. Theprecursor may also have additional polypeptides that are involved inpost-translational activity (e.g., polypeptides cleaved therefrom toleave the mature form of a protein or peptide).

The term “identical” in the context of two polynucleotide or polypeptidesequences refers to the nucleic acids or amino acids in the twosequences that are the same when aligned for maximum correspondence, asmeasured using sequence comparison or analysis algorithms describedbelow and known in the art.

The phrases “% identity” or “percent identity” or “percent sequenceidentity” or “PID” in the context of two or more nucleic acid orpolypeptide sequences refers to two or more sequences that are the sameor have a specified percentage of nucleic acid residues or amino acidresidues, respectively, that are the same, when compared and aligned formaximum similarity, as determined using a sequence comparison algorithmor by visual inspection. Percent identity may be determined usingstandard techniques known in the art. The percent amino acid identityshared by sequences of interest can be determined by aligning thesequences to directly compare the sequence information, e.g., by using aprogram such as BLAST, MUSCLE, or CLUSTAL. The BLAST algorithm isdescribed, for example, in Altschul et al., J Mol Biol, 215:403-410(1990) and Karlin et al., Proc Natl Acad Sci USA, 90:5873-5787 (1993). Apercent (%) amino acid sequence identity value is determined by thenumber of matching identical residues divided by the total number ofresidues of the “reference” sequence including any gaps created by theprogram for optimal/maximum alignment. BLAST algorithms refer to the“reference” sequence as the “query” sequence. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. These initial neighborhood word hits actas starting points to find longer HSPs containing them. The word hitsare expanded in both directions along each of the two sequences beingcompared for as far as the cumulative alignment score can be increased.Extension of the word hits is stopped when: the cumulative alignmentscore falls off by the quantity X from a maximum achieved value; thecumulative score goes to zero or below; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (See,Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992))alignments (B) of 50, expectation (E) of 10, M′5, N′-4, and a comparisonof both strands.

The BLAST algorithm then performs a statistical analysis of thesimilarity between two sequences (See e.g., Karlin and Altschul, supra).One measure of similarity provided by the BLAST algorithm is thesmallest sum probability (P(N)), which provides an indication of theprobability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a nucleic acid isconsidered similar to a lipolytic enzyme nucleic acid of this disclosureif the smallest sum probability in a comparison of the test nucleic acidto a lipolytic enzyme nucleic acid is less than about 0.1, morepreferably less than about 0.01, and most preferably less than about0.001. Where the test nucleic acid encodes a lipolytic enzymepolypeptide, it is considered similar to a specified lipolytic enzymenucleic acid if the comparison results in a smallest sum probability ofless than about 0.5, and more preferably less than about 0.2.

The CLUSTAL W algorithm is another example of a sequence alignmentalgorithm (See, Thompson et al., Nucleic Acids Res, 22:4673-4680, 1994).Default parameters for the CLUSTAL W algorithm include: Gap openingpenalty=10.0; Gap extension penalty=0.05; Protein weight matrix=BLOSUMseries; DNA weight matrix=IUB; Delay divergent sequences %=40; Gapseparation distance=8; DNA transitions weight=0.50; List hydrophilicresidues=GPSNDQEKR; Use negative matrix=OFF; Toggle Residue specificpenalties=ON; Toggle hydrophilic penalties=ON; and Toggle end gapseparation penalty=OFF. In CLUSTAL algorithms, deletions occurring ateither terminus are included. For example, a variant with a five aminoacid deletion at either terminus (or within the polypeptide) of apolypeptide of 500 amino acids would have a percent sequence identity of99% (495/500 identical residues×100) relative to the “reference”polypeptide. Such a variant would be encompassed by a variant having “atleast 99% sequence identity” to the polypeptide.

Percent sequence identity” or “% identity” or “% sequence identity or “%amino acid sequence identity” of a subject amino acid sequence to areference (i.e., query) amino acid sequence means that the subject aminoacid sequence is identical (i.e., on an amino acid-by-amino acid basis)by a specified percentage to the query amino acid sequence over acomparison length when the sequences are optimally aligned. Thus, 80%amino acid sequence identity or 80% identity with respect to two aminoacid sequences means that 80% of the amino acid residues in twooptimally aligned amino acid sequences are identical.

“Percent sequence identity” or “% identity” or “% sequence identity or“% nucleotide sequence identity” of a subject nucleic acid sequence to areference (i.e. query) nucleic acid sequence means that the subjectnucleic acid sequence is identical (i.e., on a nucleotide-by-nucleotidebasis for a polynucleotide sequence) by a specified percentage to thequery sequence over a comparison length when the sequences are optimallyaligned. Thus, 80% nucleotide sequence identity or 80% identity withrespect to two nucleic acid sequences means that 80% of the nucleotideresidues in two optimally aligned nucleic acid sequences are identical.

“Optimal alignment” or “optimally aligned” refers to the alignment oftwo (or more) sequences giving the highest percent identity score. Forexample, optimal alignment of two protein sequences can be achieved bymanually aligning the sequences such that the maximum number ofidentical amino acid residues in each sequence are aligned together orby using software programs or procedures described herein or known inthe art. Optimal alignment of two nucleic acid sequences can be achievedby manually aligning the sequences such that the maximum number ofidentical nucleotide residues in each sequence are aligned together orby using software programs or procedures described herein or known inthe art.

In some aspects, two polypeptide sequences are deemed “optimallyaligned” when they are aligned using defined parameters, such as adefined amino acid substitution matrix, gap existence penalty (alsotermed gap open penalty), and gap extension penalty, so as to achievethe highest similarity score possible for that pair of sequences. TheBLOSUM62 scoring matrix (See, Henikoff and Henikoff, supra) is oftenused as a default scoring substitution matrix in polypeptide sequencealignment algorithms (e.g., BLASTP). The gap existence penalty isimposed for the introduction of a single amino acid gap in one of thealigned sequences, and the gap extension penalty is imposed for eachresidue position in the gap. Exemplary alignment parameters employedare: BLOSUM62 scoring matrix, gap existence penalty=11, and gapextension penalty=1. The alignment score is defined by the amino acidpositions of each sequence at which the alignment begins and ends (e.g.,the alignment window), and optionally by the insertion of a gap ormultiple gaps into one or both sequences, so as to achieve the highestpossible similarity score.

Optimal alignment between two or more sequences can be determinedmanually by visual inspection or by using a computer, such as, but notlimited to for example, the BLASTP program for amino acid sequences andthe BLASTN program for nucleic acid sequences (See e.g., Altschul etal., Nucleic Acids Res. 25(17):3389-3402 (1997); See also, the NationalCenter for Biotechnology Information (NCBI) website).

A polypeptide of interest may be said to be “substantially identical” toa parent polypeptide if the polypeptide of interest comprises an aminoacid sequence having at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or at leastabout 99.5% sequence identity to the amino acid sequence of the parentpolypeptide. The percent identity between two such polypeptides can bedetermined manually by inspection of the two optimally alignedpolypeptide sequences or by using software programs or algorithms (e.g.,BLAST, ALIGN, CLUSTAL) using standard parameters. One indication thattwo polypeptides are substantially identical is that the firstpolypeptide is immunologically cross-reactive with the secondpolypeptide. Typically, polypeptides that differ by conservative aminoacid substitutions are immunologically cross-reactive. Thus, apolypeptide is substantially identical to a second polypeptide, forexample, where the two peptides differ only by a conservative amino acidsubstitution or one or more conservative amino acid substitutions.

A nucleic acid of interest may be said to be “substantially identical”to a parent nucleic acid if the nucleic acid of interest comprises anucleotide sequence having at least about 60%, at least about 65% atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or atleast about 99.5% sequence identity to the nucleotide sequence of theparent nucleic acid. The percent identity between two such nucleic acidscan be determined manually by inspection of the two optimally alignednucleic acid sequences or by using software programs or algorithms(e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. One indicationthat two nucleic acid sequences are substantially identical is that thetwo nucleic acid molecules hybridize to each other under stringentconditions (e.g., within a range of medium to high stringency).

As used herein, “homologous proteins” or “homologous lipolytic enzymes”or “homologous lipases” refers to proteins that have distinct similarityin primary, secondary, and/or tertiary structure. Protein homology canrefer to the similarity in linear amino acid sequence when proteins arealigned. Homology can be determined by amino acid sequence alignment,e.g., using a program such as BLAST, MUSCLE, or CLUSTAL. Homologoussearch of protein sequences can be done using BLASTP and PSI-BLAST fromNCBI BLAST with threshold (E-value cut-off) at 0.001. (Altschul et al.,“Gapped BLAST and PSI BLAST a new generation of protein database searchprograms”, Nucleic Acids Res, Set 1; 25(17):3389-402(1997)). The BLASTprogram uses several search parameters, most of which are set to thedefault values. The NCBI BLAST algorithm finds the most relevantsequences in terms of biological similarity but is not recommended forquery sequences of less than 20 residues (Altschul et al., Nucleic AcidsRes, 25:3389-3402, 1997 and Schaffer et al., Nucleic Acids Res,29:2994-3005, 2001). Exemplary default BLAST parameters for a nucleicacid sequence searches include: Neighboring words threshold=11; E-valuecutoff=10; Scoring Matrix=NUC.3.1 (match=1, mismatch=−3); Gap Opening=5;and Gap Extension=2. Exemplary default BLAST parameters for amino acidsequence searches include: Word size=3; E-value cutoff=10; ScoringMatrix=BLOSUM62; Gap Opening=11; and Gap extension=1. Using thisinformation, protein sequences can be grouped and/or a phylogenetic treebuilt therefrom. Amino acid sequences can be entered in a program suchas the Vector NTI Advance suite and a Guide Tree can be created usingthe Neighbor Joining (NJ) method (Saitou and Nei, Mol Biol Evol,4:406-425, 1987). The tree construction can be calculated using Kimura'scorrection for sequence distance and ignoring positions with gaps. Aprogram such as AlignX can display the calculated distance values inparenthesis following the molecule name displayed on the phylogenetictree.

When 3-dimensional structures of proteins have been determined,structurally homologous amino acid positions between two or moremolecules can be determined. When there are significant structuralsimilarities among these molecules it might be expected that introducingsubstitutions that confer improvement in one molecule at structurallyhomologous sites in another molecule could confer similar improvementsin performance and/or stability to these molecules. Structurallyhomologous amino acid positions can be identified by performing astructural alignment, which attempts to determine homology between twoor more protein structures based on their shape and three-dimensionalconformation. Structural alignment can produce a superposition of theatomic coordinate sets and a minimal root mean square deviation betweenthe structures. Examples of methods for creating structural alignmentsare the distance alignment matrix method (DALI) (Holm L, Sander C (1996)“Mapping the protein universe”. Science. 273 (5275): 595-603),combinatorial extension (CE) (Shindyalov, I. N.; Bourne P. E. (1998)“Protein structure alignment by incremental combinatorial extension (CE)of the optimal path”. Protein Engineering. 11 (9): 739-747), andSequential Structure Alignment Program (SSAP) (Taylor W R, Flores T P,Orengo C A (1994) “Multiple protein structure alignment”. Protein Sci. 3(10): 1858-70). By combining multiple sequence alignments withstructural alignments, structurally homologous amino acid positions canbe identified in molecules for which the 3-dimensional structure has notbeen determined. Examples of methods for creating multiple sequencealignment-based structural alignments are 3DCoffee (Poirot O et al(2004) “3DCoffee@igs: a web server for combining sequences andstructures into a multiple sequence alignment” Nucleic Acids Res. 2004Jul. 1; 32:W37-40), PROMALS3D (Pei J et al. (2008) “PROMALS3D: a toolfor multiple protein sequence and structure alignments.” Nucleic AcidsRes. 36(7):2295-300), and 3DM (Kuipers, R K et al (2010) “3DM:Systematic analysis of heterogeneous superfamily data to discoverprotein functionalities” Proteins 78(9):2101-13). Understanding thehomology between molecules can reveal the evolutionary history of themolecules as well as information about their function; if a newlysequenced protein is homologous to an already characterized protein,there is a strong indication of the new protein's biochemical function.The most fundamental relationship between two entities is homology; twomolecules are said to be homologous if they have been derived from acommon ancestor. Homologous molecules, or homologs, can be divided intotwo classes, paralogs and orthologs. Paralogs are homologs that arepresent within one species. Paralogs often differ in their detailedbiochemical functions. Orthologs are homologs that are present withindifferent species and have very similar or identical functions. Aprotein superfamily is the largest grouping (clade) of proteins forwhich common ancestry can be inferred. Usually this common ancestry isbased on sequence alignment and mechanistic similarity. Superfamiliestypically contain several protein families which show sequencesimilarity within the family.

A nucleic acid or polynucleotide is “isolated” when it is partially orcompletely separated from other components, including but not limited tofor example, other proteins, nucleic acids, cells, etc. Similarly, apolypeptide, protein or peptide is “isolated” when it is partially orcompletely separated from other components, including but not limited tofor example, other proteins, nucleic acids, cells, etc. On a molarbasis, an isolated species is more abundant than are other species in acomposition. For example, an isolated species may comprise at leastabout 50%, about 70%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or about 100% (on a molar basis) of all macromolecularspecies present. Preferably, the species of interest is purified toessential homogeneity (i.e., contaminant species cannot be detected inthe composition by conventional detection methods). Purity andhomogeneity can be determined using a number of techniques well known inthe art, such as agarose or polyacrylamide gel electrophoresis of aprotein or nucleic acid sample, followed by visualization upon staining.If desired, a high-resolution technique, such as high performance liquidchromatography (HPLC) or a similar means can be utilized forpurification of the material.

The term “purified” as applied to nucleic acids or polypeptidesgenerally denotes a nucleic acid or polypeptide that is essentially freefrom other components as determined by analytical techniques well knownin the art (e.g., a purified polypeptide or polynucleotide forms adiscrete band in an electrophoretic gel, chromatographic eluate, and/ora media subjected to density gradient centrifugation). For example, anucleic acid or polypeptide that gives rise to essentially one band inan electrophoretic gel is “purified.” A purified nucleic acid orpolypeptide is at least about 50% pure, usually at least about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%,about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent byweight on a molar basis). In a related sense, a composition is enrichedfor a molecule when there is a substantial increase in the concentrationof the molecule after application of a purification or enrichmenttechnique. The term “enriched” refers to a compound, polypeptide, cell,nucleic acid, amino acid, or other specified material or component thatis present in a composition at a relative or absolute concentration thatis higher than a starting composition. A substantially pure polypeptideor polynucleotide of the disclosure (e.g., substantially pure lipolyticenzyme variant or polynucleotide encoding a lipolytic enzyme variant ofthe disclosure, respectively) will typically comprise at least about55%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98, about 99%, about 99.5% or more by weight (on a molar basis) of allmacromolecular species in a particular composition.

The term “effective amount” of one or more lipolytic enzyme variantsdescribed herein or reference lipolytic enzyme(s) refers to the amountof lipolytic enzyme that achieves a desired level of enzymatic activityin a specific composition, such as but not limited to a cleaningcomposition. Such effective amounts are readily ascertained by one ofordinary skill in the art and are based on many factors, such as theparticular lipolytic enzyme used, the cleaning application, the specificcomposition of the cleaning composition, and whether a liquid or dry(e.g., granular, tablet, bar, pods) or single-unit dose composition isrequired.

The term “adjunct material” refers to any liquid, solid, or gaseousmaterial included in cleaning composition other than one or morelipolytic enzyme variants described herein, or recombinant polypeptideor active fragment thereof. In some embodiments, the cleaningcompositions of the present disclosure include one or more cleaningadjunct materials. Each cleaning adjunct material is typically selecteddepending on the particular type and form of cleaning composition (e.g.,liquid, granule, powder, bar, paste, spray, tablet, gel, foam, or othercomposition). Preferably, each cleaning adjunct material is compatiblewith the lipolytic enzyme used in the composition.

Lipolytic Enzyme Variants and Parent Enzymes

The present disclosure provides one or more lipolytic enzyme variantshaving two or more modifications as compared to a parent lipolyticenzyme.

As used herein, a “lipolytic enzyme”, “lipolytic polypeptide” and“lipolytic protein” refers to an enzyme, polypeptide, or proteinexhibiting a lipid-degrading capability such as a capability ofdegrading a triglyceride or a phospholipid. The lipolytic enzyme can be,for example, a lipase, a phospholipase, an esterase or a cutinase.Lipolytic enzymes can be lipolytic enzymes having an α/β hydrolase fold.These enzymes typically have a catalytic triad of serine, aspartic acidand histidine residues. The α/β hydrolases include lipases andcutinases. Cutinases show little, if any, interfacial activation, wherelipases often undergo a conformational change in the presence of alipid-water interface (Longhi and Cambillau (1999) Biochimica etBiophysica Acta 1441:185-96). An active fragment of a lipolytic enzymeis a portion of a lipolytic enzyme that retains a lipid degradingcapability (lipolytic activity). An active fragment retains thecatalytic triad. In one aspect, an active fragment of a parent orvariant lipase contains at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% but less than 100% of the number of the aminoacids present in the parent ot variant lipase. As used herein, lipolyticactivity can be determined according to any procedure known in the art(see, e.g., Gupta et al., Biotechnol. Appl. Biochem., 37:63-71, 2003;U.S. Pat. No. 5,990,069; and International Patent Publication No. WO96/18729A1).

In some embodiments, lipolytic enzymes of the present disclosure are a/Phydrolases. In some embodiments, lipolytic enzymes of the presentdisclosure are lipases. In some embodiments, lipolytic enzymes of thepresent disclosure are cutinases.

The term “parent”, “parent lipolytic enzyme” and “parent lipase” refersto a lipolytic enzyme to which a modification (substitution, deletion orinsertion) is made to produce the enzyme variants of the presentdisclosure. The parent may be a synthetic polypeptide, a naturallyoccurring (wild-type) polypeptide, or a variant or fragment thereof.

In some embodiments the parent lipolytic enzyme is a lipolytic enzymederived from the Genus Proteus. In one aspect the parent lipolyticenzyme comprises or consists of the amino acid sequence of SEQ ID NO: 1.SEQ ID NO:1 sets forth the amino acid sequence of the parent lipasederived from Proteus vulgaris (WP 099659650.1):

MSTTYPIVLVHGLSGFDDIVGYPYFYGIADALEKDGHKVFTASLSAFNSNEVRGEQLWEFVQKILKETKAKKVNLIGHSQGPLACRYVAAKHAKSIASVTSVNGVNHGSEIADLVRRIMRKDSVPEYIADAVMKAIGTIISTFSGNRGNPQDAIAALEALTTENVMEFNKKYPQGLPAIRGGEGKEVVNGVHYYSFGSYIQGLIAGEKGNLLDPTHAAMRVLSAFFSERENDGLVGRTSMRLGKLIKDDYAEDHLDMVNQVAGLVGRGEDIIAIYTNHANFLASKKL

The term “lypolytic enzyme variant”, “lipase variant”, “variant” and“lipolytic variant” refers to a polypeptide having lipolytic activitycomprising at least one modification, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions, when comparedto a parent lipolytic enzyme. A substitution means replacement of theamino acid occupying a position with a different amino acid; a deletionmeans removal of the amino acid occupying a position; and an insertionmeans adding an amino acid adjacent to and immediately following theamino acid occupying a position.

In some embodiments, the lipolytic enzyme variant is derived from aparent lipolytic enzyme from the Genus Proteus.

In some embodiments, the lipolytic enzyme variant is derived from aparent lipolytic enzyme having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the lipolytic enzyme variant comprises an aminoacid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequenceidentity to the amino acid sequence of SEQ ID NO: 1.

The position of an amino acid residue in a given amino acid sequence isnumbered herein using the numbering of the position of the correspondingamino acid residue of a reference lipolytic enzyme. Unless otherwiseindicated, the position of an amino acid residue in a given amino acidsequence is numbered by correspondence with the amino acid sequence ofSEQ ID NO:1. For purposes of the present disclosure, SEQ ID NO: 1 isused to determine the corresponding amino acid residue in anotherlipolytic enzyme. The amino acid sequence of SEQ ID NO:1, thus serves asa reference sequence. A given amino acid sequence, such as a lipolyticenzyme variant amino acid sequence described herein, can be aligned withthe reference lipolytic amino acid sequence of SEQ ID NO: 1 using analignment algorithm as described herein, and each amino acid residue inthe given amino acid sequence that aligns (preferably optimally aligns)with an amino acid residue in the reference lipolytic amino acidsequence of SEQ ID NO: 1 is conveniently numbered by reference to thenumerical position of that corresponding amino acid residue. Sequencealignment algorithms, such as, for example, those described herein willidentify the location where insertions or deletions occur in a subjectsequence when compared to a query sequence. In one aspect the referencelipolytic enzyme is the parent lipolytic enzyme of SEQ ID NO: 1.

The nomenclature of the amino acid substitutions of the one or morelipolytic enzyme variants described herein uses one or more of thefollowing: position; position:amino acid or amino acid substitution(s);or starting amino acid(s):position:amino acid substitution(s). Forexample, the substitution of threonine at position 4 with lysine isdesignated as “T4K”. Reference to a “position” (i.e. 4, 29, 64, etc)encompasses any starting amino acid that may be present at suchposition, and any substitution that may be present at such position.Reference to a position can be recited several forms, for example,position 003 can also be referred to as position 3. Reference to a“position: amino acid substitution(s)” (i.e. 1S/T/G, 3G, 17T, etc)encompasses any starting amino acid that may be present at such positionand the one or more amino acid(s) with which such starting amino acidmay be substituted. Reference to a starting or substituted amino acidmay be further expressed as several starting, or substituted amino acidsseparated by a foreslash (“/”). For example, D275S/K indicates position275 is substituted with serine (S) or lysine (K) and P/S197K indicatesthat starting amino acid proline (P) or serine (S) at position 197 issubstituted with lysine (K). Reference to an X as the amino acid in aposition, refers to any amino acid at the recited position.

For an amino acid deletion, the following nomenclature is used: startingamino acid: position, *. For example, the deletion of threonine atposition 4 is designated as “T4*”.

For an amino acid insertion, the following nomenclature is used:starting amino acid:position:starting amino acid:inserted amino acid.For example, the insertion of glycine after threonine at position 4 isdesignated as “T4TG”.

Multiple modifications are separated by an underscore “_” or additionmarks “+”. For example, the multiple substitution of threonine atposition 4 with lysine together with the substitution of leucine atposition 230 with arginine is designated as T4K_L203R or T4K+L203R.

The term “wild-type” lipolytic enzyme means a lipolytic enzyme expressedby a naturally occurring microorganism, such as a bacterium, yeast, orfilamentous fungus found in nature.

In one embodiment, the disclosure provides a lipolytic enzyme variant oran active fragment thereof comprising at least two amino acidmodification to a parent lipolytic enzyme, wherein the at least twoamino acid modification are at a position of the lipolytic enzymevariant selected from the group consisting of 4, 11, 19, 22, 26, 39, 53,60, 64, 67, 70, 71, 93, 95, 111, 117, 119, 120, 121, 125, 127, 131, 134,141, 142, 143, 146, 149, 154, 159, 163, 166, 170, 179, 185, 196, 199,203, 204, 205, 206, 208, 209, 212, 217, 218, 221, 224, 227, 229, 235,247, 252, 261, 262, 265, 272, 280 and 286, wherein the amino acidpositions of the variant are numbered by correspondence with the aminoacid sequence of SEQ ID NO:1, wherein the lipolytic enzyme variant hasat least 60% sequence identity to SEQ ID NO:1.

In one embodiment, the disclosure provides a lipolytic enzyme variant oran active fragment thereof comprising at least two amino acidmodifications to a parent lipolytic enzyme, wherein a first amino acidmodification is selected from the group consisting of T004K, HOI IR,I019L, Y022F, Y026C, V039I, R053Q, F060L, I064T, I064V, E067G, A070V,K071E, A093S, S095Q, I111T, R117D, R117E, R117G, R117Q, R117S, M119V,R120E, R120Q, R120S, R120V, K121E, K121S, P125E, Y127D, Y127E, A131D,A131E, A131K, A131T, K134E, K134M, S141N, T142V, F143L, N146H, N149I,I154V, A159D, E163G, M166T, K170E, K170S, I179K, K185Q, F196L, Y199H,L203C, L203K, L203N, L203R, L203S, L203T, I204V, I204Y, A205W, G206C,G206E, G206Q, K208E, K208Q, G209A, G209D, L212R, A217Y, A218D, V221T,A224S, S227D, S227V, R229A, R229C, R229H, R229P, R229Q, R229S, R229T,R229V, V23′5A, K247R, E252D, E252G, E252Q, E252V, V261A, V261L, A262C,A262M, V265A, I272K, I272P, I272V, N280K, K286Q, wherein the amino acidpositions of the variant are numbered by correspondence with the aminoacid sequence of SEQ ID NO:1 wherein the lipolytic enzyme variant has atleast 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identityto SEQ ID NO: 1.

In one embodiment, the disclosure provides a lipolytic enzyme variant oran active fragment thereof comprising at least two amino acidmodifications to a parent lipolytic enzyme, wherein a first amino acidmodification is selected from the group consisting of a K at position 4;an R at position 11; an L at position 19; an F at position 22; a C atposition 26; an I at position 39; a Q at position 53; an L at position60; a V or T at position 64; a G at position 67; a V at position 70; anE at position 71; an S at position 93; a Q at position 95; a T atposition 111; a D, E, G, Q or S at position 117; a V at position 119; anE or Q or S or V at position 120; an E or S at position 121; an E atposition 125; a D or E at position 127; a D, E, K or T at position 131;a E or M at position 134; an N at position 141; a V at position 142; anL at position 143; a H at position 146; an I at position 149; a V atposition 154; a D at position 159; a G at position 163; a T at position166; an E or S at position 170; a K at position 179; a Q at position185; a L at position 196; an H at position 199; a C, K, N, R, S ort T atposition 203; a V or Y at position 204; a W at position 205; a C, E or Qat position 206; an E or Q at position 208; an A or D at position 209; aR at position 212; a Y at position 217; a D at position 218; a T atposition 221; an S at position 224; a D or V at position 227; an A, C,H, P, Q, S, T or V at position 229; an A at position 235; an R atposition 247; a D, G, Q or V at position 252; an A or L at positon 261;a C or M at position 262; an A at position 265; a K, P or V at position272; a K at position 280; and a Q at position 286, wherein the aminoacid positions of the variant are numbered by correspondence with theamino acid sequence of SEQ ID NO: 1.

In one embodiment, the disclosure provides a lipolytic enzyme variant oran active fragment thereof comprising at least two amino acidmodification to a parent lipolytic enzyme, wherein the variant or activefragment thereof comprises amino acid modifications selected from thegroup consisting of Y022F-L203R, A131D-L203R, Y127D-L203R, A131E-L203R,L203R-I204Y, L203R-G206C, L203R-K208Q, L203R-G209D, L203R-G209A,L203R-R229Q, L203R-E252Q, L203R-V261L, L203R-A262M, L203R-A262C,L203R-I272P, Y022F-R117D-L203R, Y022F-R117Q-L203R, Y022F-A131E-L203R,Y022F-L203R-G206C, Y022F-L203R-K208Q, Y022F-L203R-G209D,Y022F-L203R-G209A, Y022F-L203R-R229Q, Y022F-L203R-R229T,Y022F-L203R-I272P, R117D-A131E-L203R, R117D-L203R-E252Q,R117Q-A131D-L203R, A131D-L203R-G206C, A131D-L203R-R229T,A131D-L203R-E252Q, R117Q-L203R-K208Q, Y127D-L203R-K208Q,A131E-L203R-K208Q, L203R-I204Y-K208Q, L203R-K208Q-G206C,L203R-K208Q-G209D, L203R-K208Q-G209A, L203R-K208Q-R229Q,L203R-K208Q-R229T, L203R-K208Q-E252Q, L203R-K208Q-V261L,L203R-K208Q-A262M, L203R-K208Q-I272P, Y127D-L203R-G209D,A131E-L203R-G209D, L203R-I204Y-G209D, L203R-G206C-G209D,L203R-G209D-R229Q, L203R-G209D-R229T, L203R-G209D-E252Q,L203R-G209D-I272P, R117Q-L203R-R229T, Y127D-L203R-R229T,A131E-L203R-R229T, L203R-I204Y-R229T, L203R-G206C-R229T,L203R-G209A-R229T, L203R-R229T-E252Q, L203R-R229T-A262M,L203R-R229T-I272P, R117Q-L203R-I272P, Y127D-L203R-I272P,A131E-L203R-I272P, L203R-I204Y-I272P, L203R-G206C-I272P,L203R-G209A-I272P, L203R-E252Q-I272P, A131D-L203R-G209D-R229T,A131D-L203R-R229T-I272P, A131D-L203S-R229T-I272P,L203R-K208Q-G209D-R229T, L203R-K208Q-R229T-I272P,L203S-K208Q-G209D-R229T, L203S-K208Q-R229T-I272P,Y022F-R117Q-A131T-F143L-L203R-G209D-R229Q,Y199H-L203R-G209D-A217Y-R229Q, L203R-G209D-A217Y-R229Q,R117Q-L203R-G209D-R229C, Y199H-A217Y,A217Y-V221T-R229C-E252Q-A262M-I272P, E252Q-A262M-I272P,Y022F-A159D-L203R-G206C-K208Q-G209D-I272P,Y022F-L203R-G206C-K208Q-G209D, P125E-L203R-K208Q-G209D,Y022F-R229C-E252Q-I272P, A159D-L203R-G209D,T004K-K071E-R117D-A131T-F143L-N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229C-N280K,T004K-K071E-R117Q-A131D-F143L-N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229C-N280K,A159D-L203R-G209D-R229H, A159D-L203R-G209D-R229C,A159D-L203R-G209D-R229V, A159D-L203R-G206E-G209D,Y022F-R117Q-A131D-L203R-R229V-E252Q,Y022F-R053Q-R117Q-E252Q-V261L-I272P, Y022F-R117Q-A131D-E252Q,Y022F-R117Q-R229V-E252Q-V261L-I272P, Y022F-R053Q-R229V-E252Q-I272P,R117Q-A131D-E252Q-I272P, Y022F-R053Q-R117Q-R229V-V261L,Y022F-R117Q-G209D-R229V-E252Q-I272P, Y022F-A131D-R229V-E252Q-I272P,Y022F-R053Q-R117Q-L203R-R229V-E252Q-V261L, Y022F-R117Q-R229V,Y022F-R117Q-A131D-R229V-E252Q,Y022F-R053Q-R117Q-L203R-R229V-E252Q-V261L-I272P,Y022F-R053Q-R117Q-L203R-R229V-E252Q-I272P,Y022F-R053Q-R117Q-L203R-R229V-E252Q,Y022F-A131D-L203R-R229V-E252Q-I272P, Y022F-R053Q-R117Q-E252Q-I272P,Y022F-R117Q-E252Q-V261L, R117Q-A131D-L203R-R229V-E252Q,Y022F-R229V-E252Q-I272P, Y022F-R117Q-R229V-I272P, Y022F-R117D,Y022F-K134E-R229Q, Y022F-R117D-I272P, R117E-A131E-I204Y-E252Q-I272K,A131E-V221T-E252Q-I272K, R117E-A131E-I204Y-G209A-V221T-R229C,R117E-A131E-R229C, R117E-V221T-R229C,Y127D-A131E-I204Y-V221T-E252Q-I272K, Y127D-G209A-V221T-R229C,R117E-G209A-V221T-E252Q, A131E-I204Y-G209A-V221T-E252Q,R117E-Y127D-A131E-I204Y-A217Y-V221T-R229C-E252Q, R117E-A131E-I272K,R117E-A131E-G209A-V221T, Y127D-A131E-A217Y-V221T-E252Q,A131E-I204Y-V221T-I272K, Y127D-G209A-V221T-E252Q-I272K,A131E-I204Y-V221T, Y127D-A131E-I204Y-E252Q,Y127D-A131E-I204Y-G209A-V221T-I272K, Y127D-A131E-G209A-E252Q-I272K,Y127D-A131E-I204Y-G209A-V221T-R229C-E252Q,Y127D-V221T-R229C-E252Q-I272K, A131E-V221T-R229C-E252Q,Y127D-I204Y-V221T-E252Q, R117E-Y127D-A131E-A217Y-V221T-E252Q-I272K,G209A-V221T-R229C-E252Q, R117E-I204Y-V221T,Y127D-I204Y-V221T-R229C-I272K, R117E-V221T-E252Q-I272K,R117E-A131E-V221T, I204Y-V221T-E252Q-I272K,R117E-Y127D-A131E-I204Y-A217Y-V221T-E252Q-I272K,A131E-G209A-V221T-R229C-I272K,Y127D-A131E-I204Y-V221T-R229C-E252Q-I272K, 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R117Q-L203R-R229S, K121S-L203R-A217Y-A224S,R117S-R120Q-L203R-R229Q, R117S-R120Q-L203R, R117S-L203R-R229A,R117S-L203R-A224S, R117Q-S227D-R229A, L203C-G209D-A217Y-S227D,R120Q-L203R-A217Y, R117S-L203R-A224S-R229C, K121S-L203C-R229A,K121E-L203N-A217Y-A224S-S227D-R229P, K121E-L203R-A217Y,A131E-G209D-A217Y, L203N-A224S-S227D-R229C, K121E-L203R-R229C,R117Q-L203R-G209D-R229A, R117Q-A224S-R229C, R117S-L203R-A224S-R229A,R117Q-K121S-L203R-S227D-R229P, R117S-Y127E-L203S-A224S-S227D,R117S-L203R-R229Q, R117Q-L203R, A131E-L203R-A224S-R229S,K121S-A131E-L203R-A224S, R117S-K121S-L203C-G206E-A217Y-A224S-S227D,R117Q-G209D, R120Q-A131E-L203R-R229C,R117Q-R120Q-Y127E-L203S-A217Y-A224S, R117Q-R120Q-L203R-A217Y-A224S,R117Q-R120Q-L203R-A217Y, R120Q-L203R-A217Y-A224S-S227D-R229S,R117Q-R120E-L203R-R229Q, R117Q-R120E-L203R-A217Y-A224S,A131E-L203R-A224S, R117S-A131E-L203N-S227D, L203S-R229C,L203S-A224S-S227D-R229A, L203R-G209D-R229C, L203R-A217Y-R229A,L203R-G206E-A217Y-S227D-R229P, L203R-A217Y-R229S,R117Q-K121E-L203R-A224S-S227D,R117S-K121S-L203C-G206E-A217Y-A224S-S227D-R229S,R117S-K121S-L203N-A217Y-R229Q, R117Q-L203S-R229C,R117Q-L203R-G206E-A224S-R229S, R117Q-R120E-L203R,R120E-G206E-A217Y-R229Q, R117S-L203S-K208E,R120Q-K121E-L203C-A217Y-R229A, A131E-L203R-A217Y-A224S-R229A,R120E-L203R-G209D-R229Q, A131E-L203R-S227D-R229P,L203R-G209D-A217Y-A224S-R229S, L203N-G206E-A217Y,R120Q-L203R-G206E-A217Y-R229Q,R117Q-K121S-L203R-G206E-A217Y-S227D-R229Q, R120E-L203R-S227D-R229A,R117S-L203R-A224S-R229S, R120Q-L203R-A224S-R229C, L203R-R229S,L203R-R229P, R117Q-K121E-L203R-R229P, R117E-R120Q-L203R-A224S,R120Q-A131E-L203C-A217Y-A224S-R229S,R117Q-R120Q-L203S-A217Y-A224S-R229C,R117Q-A131E-L203R-G206E-A217Y-A224S-S227D-R229A,R120Q-K121E-L203C-A217Y-A224S, A131E-L203N-A217Y-A224S-S227D,R117E-L203R-G209D, R120Q-L203C-G206E, L203R-G209D-S227D,R117S-L203C-G206E-A217Y-A224S, R117E-K121E-L203R,R117E-A131E-L203N-G209D-A217Y, L203C-S227D, L203S-A217Y-R229Q,R117E-A217Y-S227D, R117Q-K121S-L203R-R229Q, L203R-K208E-G209D,R117S-L203S-G206E-G209D, R117S-K121E-L203R-A224S-R229P,L203C-A217Y-S227D-R229C, R117Q-K121S-L203R,R117Q-R120Q-Y127E-L203R-A217Y-A224S, K121E-L203S-A217Y-R229P,R120Q-L203R-G206E-A217Y-A224S, I154V-K170E-A217Y, V039I-A217Y,A217Y-V265A, Y026C-K208E-A217Y, E067G-I204V-A217Y, K170E-A217Y,K134E-A217Y, A217Y-V235A, F060L-A217Y-E252G, I064T-A217Y-V261A,I111T-A217Y, M166T-A217Y, A093S-R120S-A217Y, K170S-A217Y,S141N-A217Y-A218D-E252D, S141N-A217Y-E252G, I019L-N149I-F196L-A217Y,H011R-I064V-K134M-E163G-A217Y-E252V andT004K-A070V-K071E-R117Q-A131T-F143L-N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229H-N280K.

Lipolytic Enzyme Variants with Improved Performance

The present disclosure provides one or more lipolytic enzyme variantshaving two or more modifications as compared to a parent lipolyticenzyme, including lipolytic enzyme variants having an improvedperformance compared to a parent or reference lipolytic enzyme.

As used herein, the term “improved property” (plural “improvedproperties”) and “improved performance” of a lipolytic enzyme variantrefers to a characteristic associated with a lipolytic enzyme variantthat is improved relative to the corresponding parent lipolytic enzyme.Such improved properties include, but are not limited to an improvedcleaning performance (such as improved wash performance), an increaseddetergent stability, an increased expression, a decreased (reduced)malodor, an increased thermostability, an increased calcium ion bindingstability, an increased protease stability, or any one combinationthereof.

In some aspects, the lipolytic enzyme variants of the disclosure have anincreased stability. The term “enhanced stability” or “improvedstability” in the context of an oxidation, chelator, denaturant,surfactant, thermal and/or pH stable lipolytic enzyme refers to a higherretained lipolytic activity over time as compared to a referencelipolytic enzyme, for example, a parent lipase. The stability oflipolytic enzymes of the present disclosure can be compared to thestability of a standard, for example, but not limited to, the parentlipolytic enzyme of SEQ ID NO: 1. In one aspect the lipolytic enzymevariants of the disclosure have an equal stability compared to theparent lipolytic enzyme of SEQ ID NO: 1, combined with an improvedproperty such as but not limiting to improved cleaning performance (suchas improved wash performance), an increased expression, a decreased(reduced) malodor, an increased detergent stability, an increasedthermostability, an increased calcium ion binding stability, anincreased protease stability, or any one combination thereof.

In some aspects, the lipolytic enzyme variants of the disclosure have anincreased detergent stability. As used herein, the term “enhanceddetergent stability” or “increased detergent stability” refers tolipolytic enzyme variants of the present disclosure that when present ina detergent composition, retain a higher amount of enzymatic activityover time as compared to a reference lipolytic enzyme, for example, aparent lipase. In some aspects the lipolytic enzyme variants of thedisclosure have an increased detergent stability relative to a parentlipase, such as but not limited to the parent lipase shown in SEQ IDNO:1.

In some aspects, the detergent stability of a lipolytic enzyme orvariant thereof is assayed in a liquid detergent (such as but notlimiting to a so-called heavy-duty liquid (HDL) detergent) as describedherein.

In one embodiment, the disclosure provides a lipolytic enzyme variant oran active fragment thereof comprising at least two amino acidmodifications to a parent lipolytic enzyme, wherein a first amino acidmodification is at a position of the lipolytic enzyme variant selectedfrom the group consisting of 4, 11, 19, 22, 26, 39, 53, 60, 64, 67, 70,71, 93, 95, 111, 117, 119, 120, 121, 125, 127, 131, 134, 141, 142, 143,146, 149, 154, 159, 163, 166, 170, 179, 185, 196, 199, 203, 204, 205,206, 208, 209, 212, 217, 218, 221, 224, 227, 229, 235, 247, 252, 261,262, 265, 272, 280 and 286, wherein the amino acid positions of thevariant are numbered by correspondence with the amino acid sequence ofSEQ ID NO: 1, wherein the lipolytic enzyme variant has at least 60%sequence identity to the amino acid sequence SEQ ID NO: 1, and whereinsaid lipolytic enzyme variant or active fragment thereof has an improvedperformance relative to the parent lipolytic enzyme, wherein theimproved performance is selected from the group consisting of animproved wash performance, an increased detergent stability, anincreased thermostability, and any one combination thereof.

In one embodiment, the disclosure provides a lipolytic enzyme variant oran active fragment thereof comprising at least two amino acidmodification to a parent lipolytic enzyme, wherein the variant or activefragment thereof comprises amino acid modifications selected from thegroup consisting of Y022F-L203R, A131D-L203R, Y127D-L203R, A131E-L203R,L203R-I204Y, L203R-G206C, L203R-K208Q, L203R-G209D, L203R-G209A,L203R-R229Q, L203R-E252Q, L203R-V261L, L203R-A262M, L203R-A262C,L203R-I272P, Y022F-R117D-L203R, Y022F-R117Q-L203R, Y022F-A131E-L203R,Y022F-L203R-G206C, Y022F-L203R-K208Q, Y022F-L203R-G209D,Y022F-L203R-G209A, Y022F-L203R-R229Q, Y022F-L203R-R229T,Y022F-L203R-I272P, R117D-A131E-L203R, R117D-L203R-E252Q,R117Q-A131D-L203R, A131D-L203R-G206C, A131D-L203R-R229T,A131D-L203R-E252Q, R117Q-L203R-K208Q, Y127D-L203R-K208Q,A131E-L203R-K208Q, L203R-I204Y-K208Q, L203R-K208Q-G206C,L203R-K208Q-G209D, L203R-K208Q-G209A, L203R-K208Q-R229Q,L203R-K208Q-R229T, L203R-K208Q-E252Q, L203R-K208Q-V261L,L203R-K208Q-A262M, L203R-K208Q-I272P, Y127D-L203R-G209D,A131E-L203R-G209D, L203R-I204Y-G209D, L203R-G206C-G209D,L203R-G209D-R229Q, L203R-G209D-R229T, L203R-G209D-E252Q,L203R-G209D-I272P, R117Q-L203R-R229T, Y127D-L203R-R229T,A131E-L203R-R229T, L203R-I204Y-R229T, L203R-G206C-R229T,L203R-G209A-R229T, L203R-R229T-E252Q, L203R-R229T-A262M,L203R-R229T-I272P, R117Q-L203R-I272P, Y127D-L203R-I272P,A131E-L203R-I272P, L203R-I204Y-I272P, L203R-G206C-I272P,L203R-G209A-I272P, L203R-E252Q-I272P, A131D-L203R-G209D-R229T,A131D-L203R-R229T-I272P, A131D-L203S-R229T-I272P,L203R-K208Q-G209D-R229T, L203R-K208Q-R229T-I272P,L203S-K208Q-G209D-R229T, L203S-K208Q-R229T-I272P,Y022F-R117Q-A131T-F143L-L203R-G209D-R229Q,Y199H-L203R-G209D-A217Y-R229Q, L203R-G209D-A217Y-R229Q,R117Q-L203R-G209D-R229C, Y199H-A217Y,A217Y-V221T-R229C-E252Q-A262M-I272P, E252Q-A262M-I272P,Y022F-A159D-L203R-G206C-K208Q-G209D-I272P,Y022F-L203R-G206C-K208Q-G209D, P125E-L203R-K208Q-G209D,Y022F-R229C-E252Q-I272P, A159D-L203R-G209D,T004K-K071E-R117D-A131T-F143L-N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229C-N280K,T004K-K071E-R117Q-A131D-F143L-N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229C-N280K,A159D-L203R-G209D-R229H, A159D-L203R-G209D-R229C,A159D-L203R-G209D-R229V, A159D-L203R-G206E-G209D,Y022F-R117Q-A131D-L203R-R229V-E252Q,Y022F-R053Q-R117Q-E252Q-V261L-I272P, Y022F-R117Q-A131D-E252Q,Y022F-R117Q-R229V-E252Q-V261L-I272P, Y022F-R053Q-R229V-E252Q-I272P,R117Q-A131D-E252Q-I272P, Y022F-R053Q-R117Q-R229V-V261L,Y022F-R117Q-G209D-R229V-E252Q-I272P, Y022F-A131D-R229V-E252Q-I272P,Y022F-R053Q-R117Q-L203R-R229V-E252Q-V261L, Y022F-R117Q-R229V,Y022F-R117Q-A131D-R229V-E252Q,Y022F-R053Q-R117Q-L203R-R229V-E252Q-V261L-I272P,Y022F-R053Q-R117Q-L203R-R229V-E252Q-I272P,Y022F-R053Q-R117Q-L203R-R229V-E252Q,Y022F-A131D-L203R-R229V-E252Q-I272P, Y022F-R053Q-R117Q-E252Q-I272P,Y022F-R117Q-E252Q-V261L, R117Q-A131D-L203R-R229V-E252Q,Y022F-R229V-E252Q-I272P, Y022F-R117Q-R229V-I272P, Y022F-R117D,Y022F-K134E-R229Q, Y022F-R117D-I272P, R117E-A131E-I204Y-E252Q-I272K,A131E-V221T-E252Q-I272K, R117E-A131E-I204Y-G209A-V221T-R229C,R117E-A131E-R229C, R117E-V221T-R229C,Y127D-A131E-I204Y-V221T-E252Q-I272K, Y127D-G209A-V221T-R229C,R117E-G209A-V221T-E252Q, A131E-I204Y-G209A-V221T-E252Q,R117E-Y127D-A131E-I204Y-A217Y-V221T-R229C-E252Q, R117E-A131E-I272K,R117E-A131E-G209A-V221T, Y127D-A131E-A217Y-V221T-E252Q,A131E-I204Y-V221T-I272K, Y127D-G209A-V221T-E252Q-I272K,A131E-I204Y-V221T, Y127D-A131E-I204Y-E252Q,Y127D-A131E-I204Y-G209A-V221T-I272K, Y127D-A131E-G209A-E252Q-I272K,Y127D-A131E-I204Y-G209A-V221T-R229C-E252Q,Y127D-V221T-R229C-E252Q-I272K, A131E-V221T-R229C-E252Q,Y127D-I204Y-V221T-E252Q, R117E-Y127D-A131E-A217Y-V221T-E252Q-I272K,G209A-V221T-R229C-E252Q, R117E-I204Y-V221T,Y127D-I204Y-V221T-R229C-I272K, R117E-V221T-E252Q-I272K,R117E-A131E-V221T, I204Y-V221T-E252Q-I272K,R117E-Y127D-A131E-I204Y-A217Y-V221T-E252Q-I272K,A131E-G209A-V221T-R229C-I272K,Y127D-A131E-I204Y-V221T-R229C-E252Q-I272K, R117E-V221T-I272K,Y127D-A131E-I204Y-I272K, A131E-E252Q,R117E-A131E-I204Y-G209A-V221T-E252Q-I272K,Y127D-A131E-G209A-R229C-E252Q, A131E-G209A-V221T-R229C,R117E-A131E-I204Y-G209A-V221T-I272K, R117E-A131E-I204Y-E252Q,R117E-A131E-I204Y-V221T-R229C-E252Q, A131E-G209A-V221T-I272K,Y127D-E252Q, R117E-A131E-I204Y-G209A-V221T-R229C-E252Q-I272K,R117E-V221T-R229C-E252Q, Y127D-A131E-I204Y-G209A-R229C-E252Q-I272K,R117E-A131E-I204Y-I272K, R117E-A131E-I204Y-V221T-R229C-I272K,R117E-A131E-A217Y-V221T-E252Q, I204Y-G209A-V221T-E252Q-I272K,R117E-Y127D-I204Y-E252Q, I204Y-G209A-V221T,R117E-Y127D-A131E-G209A-E252Q-I272K, R117E-A131E-R229C-E252Q,Y127D-A131E-I204Y-G209A-V221T-R229C-I272K,Y127D-A131E-I204Y-G209A-V221T-R229C,Y127D-A131E-G209A-R229C-E252Q-I272K,A131E-I204Y-G209A-V221T-E252Q-I272K, Y127D-A131E-I204Y-E252Q-I272K,Y127D-A131E-I204Y, R117E-I204Y-G209A-V221T-E252Q,R117E-A131E-G209A-V221T-R229C-E252Q, A131E-I204Y-G209A-V221T-R229C,R117E-V221T-R229C-I272K, Y127D-I204Y-G209A-E252Q-I272K,G209A-V221T-R229C-E252Q-I272K, Y127D-I204Y-V221T-R229C,Y127D-I204Y-G209A-V221T-I272K, R117E-A131E-V221T-I272K,Y127D-I204Y-G209A-V221T-E252Q, L203S-E252Q, L203S-E252Q-I272P,M119V-L203S-E252Q, L203S-S227V-E252Q, M119V-L203S-S227V-E252Q-I272P,M119V-L203S-S227V-E252Q-I272V, R117Q-M119V-L203S-S227V-E252Q-I272P,R117Q-M119V-L203S-S227V-E252Q-I272V,T142V-K185Q-L203S-S227V-E252Q-I272V-K286Q,M119V-K185Q-L203S-S227V-E252Q-I272V-K286Q,M119V-T142V-L203S-S227V-E252Q-I272V-K286Q,M119V-T142V-K185Q-S227V-E252Q-I272V-K286Q,M119V-T142V-K185Q-L203S-S227V-I272V-K286Q,M119V-T142V-K185Q-L203S-S227V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227D-R229V-E252Q,M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q-I272V,R117Q-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q-I272V,R117Q-M119V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-I204Y-G206Q-S227V-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-S227V-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227D-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q-I272P,A131E-L203K-G206Q-R229V-E252Q-I272V,A131E-L203T-G206Q-R229V-E252Q-I272V, A131E-L203K-R229V-E252Q-I272V,A131E-L203K-G206Q-E252Q-I272V, L203T-G206Q-R229V-E252Q-I272V,A131E-L203T-G206Q-R229V-I272V,S095Q-A131E-L203K-G206Q-R229V-K247R-E252Q-I272V,S095Q-L203K-G206Q-R229V-K247R-E252Q-I272V,S095Q-A131E-L203T-G206Q-R229V-K247R-E252Q-I272V,S095Q-A131E-G206Q-R229V-K247R-E252Q-I272V,S095Q-A131E-L203K-R229V-K247R-E252Q-I272V,S095Q-A131E-L203K-G206Q-K247R-E252Q-I272V,S095Q-A131E-L203K-G206Q-R229V-E252Q-I272V,S095Q-A131E-L203K-G206Q-R229V-K247R-I272V,S095Q-A131E-L203K-G206Q-R229V-K247R-E252Q,A131E-L203S-G206Q-R229V-I272P, L203S-G206Q-R229V-I272P,A131E-L203S-R229V-I272P, A131E-L203S-G206Q-I272P,A131E-L203S-G206Q-R229V, R117Q-L203T-A224S-E252Q,R117Q-A131E-L203S-I204Y-S227V-R229Q-E252Q-I272P,A131E-L203S-I204Y-S227V-R229Q-E252Q-I272P,R117Q-L203S-I204Y-S227V-R229Q-E252Q-I272P,R117Q-A131E-I204Y-S227V-R229Q-E252Q-I272P,R117Q-A131E-L203S-S227V-R229Q-E252Q-I272P,R117Q-A131E-L203S-I204Y-R229Q-E252Q-I272P,R117Q-A131E-L203S-I204Y-S227V-R229Q-I272P,R117Q-A131E-L203S-I204Y-S227V-R229Q-E252Q,A131E-L203S-G206Q-A217Y-R229V-I272P,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-A217Y-S227D-R229V-E252Q,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-A217Y-S227D-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-A217Y-S227V-R229V-E252Q-I272P,R117Q-M119V-R120V-A131E-L203T-I204Y-A205W-G206Q-S227D-R229V-E252Q,R117Q-M119V-R120V-A131E-L203T-I204Y-A205W-G206Q-S227D-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-A205W-G206Q-S227V-R229V-E252Q-I272P,R117E-Y127D-A131E-I204Y-A205W-A217Y-V221T-R229C-E252Q,R117G-Y127D-A131E-I204Y-A217Y-V221T-R229Q-E252Q,R117G-Y127D-A131E-I204Y-A205W-A217Y-V221T-R229Q-E252Q,R117G-Y127D-A131E-A217Y-V221T-E252Q-I272K,R117G-Y127D-A131E-A205W-A217Y-V221T-E252Q-I272K,R117G-A131E-I204Y-A217Y-V221T-R229Q-E252Q,R117G-A131E-I204Y-A205W-A217Y-V221T-R229Q-E252Q,R117G-A131E-I204Y-G209A-A217Y-V221T-R229Q-E252Q-I272K,R117G-A131E-I204Y-A205W-G209A-A217Y-V221T-R229Q-E252Q-I272K,R117E-A131E-A205W-A217Y-V221T-E252Q,Y127D-A131E-I204Y-A205W-G209A-A217Y-V221T-R229Q, R117E-A131E-A217Y,A131E-A217Y, L203R-A217Y, L203R-G209D-A217Y, G209D-A217Y,Y022F-R117Q-L203R-G209D-A217Y,Y022F-A159D-L203R-G206C-K208Q-G209D-A217Y-I272P,R117E-A131E-I204Y-G209A-A217Y, R117E-A131E-I204Y-G209A-A217Y-V221T,R117E-A131E-I204Y-G209A-A217Y-V221T-R229C-E252Q-I272K,R117E-A131E-F143L-I204Y-G209A-A217Y-V221T-R229C-E252Q-I272K,Y022F-L203R-G206C-K208Q-G209D-A217Y, Y022F-R117E-A131E-L203R-G209D,Y022F-R117E-A131E-L203R-G209D-A217Y, R117E-A131E-L203R-G209D,R117E-A131E-L203R-G209D-A217Y, R117E-A131E-L203R-I204Y-G209A-V221T,R117E-A131E-L203R-I204Y-G209A-A217Y-V221T, A217Y-E252Q,R117E-A131E-I204Y-G209A-A217Y-E252Q,Y022F-R053Q-R117Q-F143L-L203R-G206C-K208Q-R229V-E252Q,Y022F-R117D-F143L-I272P, Y022F-R117E-A131E-A217Y-V221T,Y022F-R117E-A131E-F143L-A217Y-V221T, A131E-L203R-A217Y-I272P,A131E-F143L-L203R-A217Y-I272P, Y022F-R053Q-F143L-R229V-E252Q-I272P,R117E-A131E-A217Y-V221T-E252Q-I272K,R117E-Y127D-A131E-A217Y-E252Q-I272K,R117E-Y127D-A131E-A217Y-V221T-E252Q,R117E-Y127D-A131E-A217Y-V221T-I272K,R117E-Y127D-A131E-I204Y-G209A-A217Y-V221T-E252Q-I272K,R117E-Y127D-A131E-F143L-I204Y-G209A-A217Y-V221T-R229C-E252Q-I272K,Y127D-A217Y, R117E-A131E-A217Y-V221T,Y022F-A159D-L203R-G209D-A217Y-V221T-I272K,Y022F-P125E-A131E-I179K-L203R-G209D-A217Y-V221T-R229C-I272K,A131E-A217Y-V221T, A131E-G209D-V221T-I272K,A131E-L203R-G209D-A217Y-V221T,A131E-L203R-G209D-A217Y-V221T-R229C-I272K,R117E-Y127D-L203S-A217Y-E252Q, Y022F-A217Y-E252Q,Y022F-L203R-G206C-G209D-A217Y-E252Q,R117Q-A131E-L203S-G209D-A217Y-E252Q, R117Q-Y127D-A217Y-E252Q,A131D-R229V-E252Q-I272P, Y022F-A131D-A217Y-R229V-E252Q-I272P,Y022F-A131E-R229V-E252Q-I272P, Y022F-A131D-R229V-E252Q-I272K,Y022F-A131E-R229V-E252Q-I272K,R117E-Y127D-A131D-A217Y-V221T-E252Q-I272P,R117E-Y127D-A131D-A217Y-V221T-R229C-E252Q-I272P,R117E-Y127D-A131D-A217Y-V221T-R229V-E252Q-I272P,Y127D-A131E-A217Y-V221T-R229V-E252Q-I272K,Y127D-A131E-A217Y-V221T-R229C-E252Q-I272K,Y127D-A131E-A159D-A217Y-V221T-R229C-E252Q-I272K, L203R-R229V-E252Q,L203R-G209D-A217Y-E252Q, L203R-G209A-A217Y-E252Q, L203R-A217Y-R229V,L203R-G209D-A217Y-R229Q-E252Q, L203R-G209D-A217Y-R229V-E252Q,A159D-L203R-G209D-A217Y-E252Q, A159D-L203R-G209D-A217Y,A159D-L203R-G209D-A217Y-I272K, Y022F-L203R-G209D-A217Y,Y022F-L203R-G209D-A217Y-E252Q,Y127D-A131E-I204Y-A217Y-V221T-R229C-E252Q-I272K,Y127D-A131E-L203R-A217Y-V221T-R229C-E252Q-I272K,L203S-G209D-A217Y-R229A, L203N-A217Y-A224S-R229A,R117Q-L203S-A217Y-A224S-S227D-R229S, R117Q-K121S-L203R-R229C,R117Q-A131E-L203R, L203C-A217Y-A224S-R229P,R117Q-K121S-L203C-G206E-A217Y-S227D-R229S,A131E-L203R-G209D-A224S-R229P, R120Q-A131E-L203R,R117Q-L203R-A217Y-A224S-S227D-R229A, A217Y-A224S-R229A,L203R-S227D-R229Q, A131E-L203R-G209D-R229S, K121S-L203R,R117S-L203C-G206E-A217Y-R229Q, R117E-L203R-G209D-A224S-R229S,R120Q-L203C-G209D-R229S, L203R-K208E-G209D-A224S,R117S-R120Q-K121S-L203R-A217Y-A224S-R229C, L203R-A224S,R117Q-A131E-L203C-A217Y-S227D-R229P, R117S-L203S-A224S,Y127E-L203S-A217Y-R229C, R117Q-L203R-G206E-G209D-A217Y-S227D,R117S-K121S-L203R-R229P, R117S-R120Q-A131E, R117E-G209D,R117S-R120Q-Y127E-L203R-G206E-A217Y, L203R-A217Y-A224S-R229Q,R120Q-G206E-G209D-A217Y-A224S, R117E-R120Q-L203R-G209D-A217Y-A224S,A131E-G209D-A217Y-A224S-R229A, G209D-A224S,R117S-Y127E-A217Y-A224S-S227D-R229C, K121E-L203N-A217Y-R229A,Y127E-A217Y-A224S-R229S, R117S-A131E-L203S-R229S, L203R-K208E-R229A,R117Q-A131K-L203R-A224S, L203R-R229A, R120E-L203R-R229P, R120Q-A217Y,R120Q-L203N-A217Y-A224S-R229S, L203R-A217Y-A224S-R229A,L203S-K208E-G209D-A217Y-A224S-S227D-R229S, R117S-G209D-A217Y,L203S-G209D-S227D, A131E-L203R-A224S-R229A, L203R-A224S-R229P,Y127E-L203R-A217Y-R229C, R117Q-L203R-R229S, K121S-L203R-A217Y-A224S,R117S-R120Q-L203R-R229Q, R117S-R120Q-L203R, R117S-L203R-R229A,R117S-L203R-A224S, R117Q-S227D-R229A, L203C-G209D-A217Y-S227D,R120Q-L203R-A217Y, R117S-L203R-A224S-R229C, K121S-L203C-R229A,K121E-L203N-A217Y-A224S-S227D-R229P, K121E-L203R-A217Y,A131E-G209D-A217Y, L203N-A224S-S227D-R229C, K121E-L203R-R229C,R117Q-L203R-G209D-R229A, R117Q-A224S-R229C, R117S-L203R-A224S-R229A,R117Q-K121S-L203R-S227D-R229P, R117S-Y127E-L203S-A224S-S227D,R117S-L203R-R229Q, R117Q-L203R, A131E-L203R-A224S-R229S,K121S-A131E-L203R-A224S, R117S-K121S-L203C-G206E-A217Y-A224S-S227D,R117Q-G209D, R120Q-A131E-L203R-R229C,R117Q-R120Q-Y127E-L203S-A217Y-A224S, R117Q-R120Q-L203R-A217Y-A224S,R117Q-R120Q-L203R-A217Y, R120Q-L203R-A217Y-A224S-S227D-R229S,R117Q-R120E-L203R-R229Q, R117Q-R120E-L203R-A217Y-A224S,A131E-L203R-A224S, R117S-A131E-L203N-S227D, L203S-R229C,L203S-A224S-S227D-R229A, L203R-G209D-R229C, L203R-A217Y-R229A,L203R-G206E-A217Y-S227D-R229P, L203R-A217Y-R229S,R117Q-K121E-L203R-A224S-S227D,R117S-K121S-L203C-G206E-A217Y-A224S-S227D-R229S,R117S-K121S-L203N-A217Y-R229Q, R117Q-L203S-R229C,R117Q-L203R-G206E-A224S-R229S, R117Q-R120E-L203R,R120E-G206E-A217Y-R229Q, R117S-L203S-K208E,R120Q-K121E-L203C-A217Y-R229A, A131E-L203R-A217Y-A224S-R229A,R120E-L203R-G209D-R229Q, A131E-L203R-S227D-R229P,L203R-G209D-A217Y-A224S-R229S, L203N-G206E-A217Y,R120Q-L203R-G206E-A217Y-R229Q,R117Q-K121S-L203R-G206E-A217Y-S227D-R229Q, R120E-L203R-S227D-R229A,R117S-L203R-A224S-R229S, R120Q-L203R-A224S-R229C, L203R-R229S,L203R-R229P, R117Q-K121E-L203R-R229P, R117E-R120Q-L203R-A224S,R120Q-A131E-L203C-A217Y-A224S-R229S,R117Q-R120Q-L203S-A217Y-A224S-R229C,R117Q-A131E-L203R-G206E-A217Y-A224S-S227D-R229A,R120Q-K121E-L203C-A217Y-A224S, A131E-L203N-A217Y-A224S-S227D,R117E-L203R-G209D, R120Q-L203C-G206E, L203R-G209D-S227D,R117S-L203C-G206E-A217Y-A224S, R117E-K121E-L203R,R117E-A131E-L203N-G209D-A217Y, L203C-S227D, L203S-A217Y-R229Q,R117E-A217Y-S227D, R117Q-K121S-L203R-R229Q, L203R-K208E-G209D,R117S-L203S-G206E-G209D, R117S-K121E-L203R-A224S-R229P,L203C-A217Y-S227D-R229C, R117Q-K121S-L203R,R117Q-R120Q-Y127E-L203R-A217Y-A224S, K121E-L203S-A217Y-R229P,R120Q-L203R-G206E-A217Y-A224S, I154V-K170E-A217Y, V039I-A217Y,A217Y-V265A, Y026C-K208E-A217Y, E067G-I204V-A217Y, K170E-A217Y,K134E-A217Y, A217Y-V235A, F060L-A217Y-E252G, 1064T-A217Y-V261A,111T-A217Y, M166T-A217Y, A093S-R120S-A217Y, K170S-A217Y,S141N-A217Y-A218D-E252D, S141N-A217Y-E252G, I019L-N149I-F196L-A217Y,H011R-1064V-K134M-E163G-A217Y-E252V andT004K-A070V-K071E-R117Q-A131T-F143L-N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229H-N280K,wherein the amino acid positions of the variant are numbered bycorrespondence with the amino acid sequence of SEQ ID NO: 1, wherein thelipolytic enzyme variant has at least 60% sequence identity to the aminoacid sequence SEQ ID NO: 1, and wherein said lipolytic enzyme variant oractive fragment thereof has an increased detergent stability compared tothe parent lipolytic enzyme.

In one embodiment, the disclosure provides a lipolytic enzyme variant oran active fragment thereof comprising at least two amino acidmodifications to a parent lipolytic enzyme, wherein a first amino acidmodification is selected from the group consisting of a K at position 4;an R at position 11; an L at position 19; an F at position 22; a C atposition 26; an I at position 39; a Q at position 53; an L at position60; a V or T at position 64; a G at position 67; a V at position 70; anE at position 71; an S at position 93; a Q at position 95; a T atposition 111; a D, E, G, Q or S at position 117; a V at position 119; anE or Q or S or V at position 120; an E or S at position 121; an E atposition 125; a D or E at position 127; a D, E, K or T at position 131;a E or M at position 134; an N at position 141; a V at position 142; anL at position 143; a H at position 146; an I at position 149; a V atposition 154; a D at position 159; a G at position 163; a T at position166; an E or S at position 170; a K at position 179; a Q at position185; a L at position 196; an H at position 199; a C, K, N, R, S ort T atposition 203; a V or Y at position 204; a W at position 205; a C, E or Qat position 206; an E or Q at position 208; an A or D at position 209; aR at position 212; a Y at position 217; a D at position 218; a T atposition 221; an S at position 224; a D or V at position 227; an A, C,H, P, Q, S, T or V at position 229; an A at position 235; an R atposition 247; a D, G, Q or V at position 252; an A or L at positon 261;a C or M at position 262; an A at position 265; a K, P or V at position272; a K at position 280; and a Q at position 286, wherein the aminoacid positions of the variant are numbered by correspondence with theamino acid sequence of SEQ ID NO: 1, wherein the amino acid positions ofthe variant are numbered by correspondence with the amino acid sequenceof SEQ ID NO: 1, wherein the lipolytic enzyme variant has at least 60%sequence identity to the amino acid sequence SEQ ID NO: 1, and whereinsaid lipolytic enzyme variant or active fragment thereof has anincreased detergent stability compared to the parent lipolytic enzyme.

In some aspects, the lipolytic enzyme variants of the disclosure have anincreased thermostability. In one aspect the lipolytic enzyme variantsof the disclosure have an increased thermostability relative to a parentlipase, such as but not limited to the parent lipase shown in SEQ ID NO:1.

As used herein “thermal stability” and “thermostability” and“thermostable” refer to lipolytic enzyme variants of the presentdisclosure that retain a specified amount of enzymatic activity afterexposure to an identified temperature, often over a given period of timeunder conditions prevailing during the lipolytic, hydrolyzing, cleaningor other process disclosed herein, for example while exposed to alteredtemperatures. Altered temperatures include increased or decreasedtemperatures. In some embodiments, the lipolytic enzyme variant retainsat least about 40%, about 50%, about 60%, about 70%, about 80%, about85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%,or about 99% lipolytic activity after exposure to altered temperaturesover a given time period, for example, at least about 20 minutes, atleast about 30 minutes, at least about 40 minutes, at least about 50minutes, at least about 60 minutes, about 90 minutes, about 120 minutes,about 180 minutes, about 240 minutes, about 300 minutes, about 360minutes, about 420 minutes, about 480 minutes, about 540 minutes, about600 minutes, about 660 minutes, about 720 minutes, about 780 minutes,about 840 minutes, about 900 minutes, about 960 minutes, about 1020minutes, about 1080 minutes, about 1140 minutes, or about 1200 minutes.

In one aspect the identified altered temperature is at least about 45°C.

In one aspect, the lipolytic enzyme variant or active fragment thereofof the disclosure, has a thermostability performance index(PI(thermostability)) relative to the parent lipolytic enzyme that isgreater than 1.0.

In some aspects, the lipolytic enzyme variants of the disclosure have animproved wash performance. The term “wash performance” and “cleaningperformance” refers to a cleaning performance achieved by lipolyticenzyme or reference lipase under conditions prevailing during thelipolytic, hydrolyzing, cleaning, or other process of the disclosure. Insome embodiments, cleaning performance of a lipolytic enzyme orreference lipase may be determined by using various assays for cleaningone or more lipase-sensitive stains on an item or surface (e.g., beeffat, butterfat, lard, margarine, vegetable oil, bacon grease, sebum).Cleaning performance of one or more lipolytic enzyme variants describedherein or reference lipase can be determined by subjecting the stain onthe item or surface to standard wash condition(s) and assessing thedegree to which the stain is removed by using various chromatographic,spectrophotometric, or other quantitative methodologies.

In one aspect, the lipolytic variant has an improved wash performance,wherein the the lipolytic variant has a wash performance index(PI(wash)) relative to a parent lipolytic enzyme that is greater than1.0. In some aspects, the wash performance is compared under relevantwashing conditions. In some test systems, other relevant factors, suchas detergent composition, suds concentration, water hardness, washingmechanics, time, pH, and/or temperature, can be controlled in such a waythat condition(s) typical for household application in a certain marketsegment (e.g., hand or manual dishwashing, automatic dishwashing,dishware cleaning, tableware cleaning, fabric cleaning, etc.) areimitated.

In one embodiment, the disclosure provides a lipolytic enzyme variantlipolytic enzyme variant or an active fragment thereof comprising atleast two amino acid modifications to a parent lipolytic enzyme, whereinthe variant or active fragment thereof comprises amino acidmodifications selected from the group consisting of L203R-G206C-I272P,P125E-L203R-K208Q-G209D, L203R-E252Q-I272P,Y022F-R117Q-A131T-F143L-L203R-G209D-R229Q, L203S-K208Q-G209D-R229T,R117Q-L203R-G209D-R229C, L203R-G209A-I272P, A131D-L203S-R229T-I272P,R117E-A131E-I204Y-E252Q-I272K, Y127D-A131E-A217Y-V221T-E252Q,L203R-I204Y-I272P, Y127D-I204Y-V221T-R229C-I272K,Y127D-A131E-I204Y-G209A-V221T-I272K,R117E-A131E-I204Y-G209A-V221T-E252Q-I272K,Y127D-V221T-R229C-E252Q-I272K, L203S-K208Q-R229T-I272P,Y127D-L203R-I272P, Y127D-I204Y-V221T-R229C,R117E-A131E-I204Y-G209A-V221T-I272K,R117E-A131E-I204Y-G209A-V221T-R229C, R117E-A131E-I204Y-I272K,A131E-L203R-K208Q, R117Q-L203R-K208Q, L203R-R229T-A262M,R117E-V221T-R229C-E252Q, A131E-G209A-V221T-R229C,G209A-V221T-R229C-E252Q, R117E-A131E-G209A-V221T-R229C-E252Q,R117E-A131E-I204Y-V221T-R229C-E252Q, R117E-I204Y-G209A-V221T-E252Q,A131E-L203R-G209D,T004K-A070V-K071E-R117Q-A131T-F143L-N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229H-N280K,Y127D-A131E-I204Y-G209A-R229C-E252Q-I272K, L203R-R229T-E252Q,Y127D-A131E-I204Y-V221T-R229C-E252Q-I272K, L203R-G209D-I272P,L203R-G209D-R229Q, L203R-K208Q, L203R-K208Q-E252Q, R117Q-L203R-R229T,Y022F-R117Q-R229V-I272P, R117E-A131E-I204Y-V221T-R229C-I272K,A131D-L203R-E252Q, L203R-K208Q-R229T-I272P, R117Q-A131D-E252Q-I272P,R117E-A131E-I204Y-G209A-V221T-R229C-E252Q-I272K,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-E252Q-I272V,Y022F-R053Q-R117Q-L203R-R229V-E252Q, R117E-A131E-R229C,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q-I272P,Y127D-I204Y-G209A-E252Q-I272K, L203R-G209D, L203R-I272P,L203R-K208Q-A262M, R117E-A131E-R229C-E252Q,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-A217Y-S227D-R229V-E252Q-I272V,L203R-R229T-I272P,R117G-A131E-I204Y-A205W-G209A-A217Y-V221T-R229Q-E252Q-I272K,R117Q-A131E-L203S-I204Y-S227V-R229Q-E252Q-I272P,Y022F-R053Q-R117Q-E252Q-I272P, Y022F-L203R-R229Q,Y022F-R117Q-E252Q-V261L, L203R-G209D-E252Q, Y127D-G209A-V221T-R229C,L203R-R229Q, R117Q-L203R-I272P, L203R-K208Q-G209D-R229T,R117S-L203R-A224S-R229S, L203R-G206C-G209D, R117E-A131E-I204Y-E252Q,Y127D-A131E-I204Y-G209A-V221T-R229C, L203N-A217Y-A224S-R229A,L203R-I204Y, Y022F-L203R-R229T, Y127D-A131E-G209A-R229C-E252Q-I272K,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-A217Y-S227V-R229V-E252Q-I272P,Y022F-R117Q-G209D-R229V-E252Q-I272P,R117Q-M119V-R120V-A131E-L203T-I204Y-A205W-G206Q-S227V-R229V-E252Q-I272P,R117S-L203C-G206E-A217Y-R229Q, Y022F-L203R-I272P andR117Q-L203S-A217Y-A224S-S227D-R229S, wherein the amino acid positions ofthe variant are numbered by correspondence with the amino acid sequenceof SEQ ID NO: 1, wherein the lipolytic enzyme variant has at least 60%sequence identity to the amino acid sequence SEQ ID NO: 1, and whereinsaid lipolytic enzyme variant or active fragment thereof has an improvedwash performance relative to the wash performance of said parentlipolytic enzyme

In some embodiments, one or more compositions described herein comprisesone or more lipolytic enzyme variants described herein as useful oreffective for cleaning a surface in need of oily-stain removal.

In some embodiments, one or more lipolytic enzyme variants describedherein cleans at low temperatures, also referred to as coldtemperatures. In other embodiments, one or more compositions describedherein cleans at low temperatures. In one aspect, the lipolytic varianthas an improved wash performance, wherein the improved wash performanceis an improved wash performance at a low temperature. Low temperaturesinclude cleaning temperatures of 10° C., 11° C., 12° C., 13° C., 4C, 15°C., 16° C., 17° C., 18° C., 19° C., 20° C., 21C, 22° C., 23° C., 24° C.,25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C.,34° C., 35° C., 36° C., 37° C., 38° C., 39° C. and up to 40° C. Lowtemperatures include cleaning or washing temperatures from about 10° C.to about 40° C., or from about 20° C. to about 30° C., or from about 15°C. to about 25° C., as well as all other combinations within the rangeof about 15° C. to about 35° C., and all ranges within 10° C. to 40° C.

In one aspect, the lipolytic variant has an improved wash performance,wherein the improved wash performance is an improved wash performanceduring cold water washing. Cold water washing of the present disclosureutilizes cold water detergent comprising one or more lipolytic enzymevariants described herein, suitable for washing at temperatures fromabout 10° C. to about 40° C., or from about 20° C. to about 30° C., orfrom about 15° C. to about 25° C., as well as all other combinationswithin the range of about 15° C. to about 35° C., and all ranges within10° C. to 40° C.

In one aspect, the lipolytic variant has an improved wash performance,wherein the improved wash performance is at a cleaning temperature of40° C.

In some aspects, the lipolytic enzyme variants of the disclosure have adecreased (reduced) malodor performance, where less malodor is producedrelative to a reference polypeptide. In some aspects, the lipolyticenzyme variants of the disclosure have an increased calcium ion bindingstability. In one aspect, the lipolytic enzyme variant or activefragment thereof of the disclosure, has a calcium ion binding stabilityindex (PI(calcium ion binding stability)) relative to the parentlipolytic enzyme that is greater than 1.0.

An increased calcium ion binding stability may occur when the lipolyticenzyme retains the same level of activity when exposed to a reduced orlow levels of Ca ion, when compared to a control. The term “reduced orlow levels of Ca ion” means that the concentration of Ca ion in asolution has been reduced or lowered as compared to a control solution.Such reduced or low levels of Ca ion may be obtained by adding an agentthat depletes a part or all Ca ion from the solution, including Ca ionthat may be bound to the lipolytic enzyme present in the solution.

An increased calcium ion binding stability may occur when the lipolyticenzyme retains the same level of activity when exposed to EDTA(ethylenediaminetetraacetic acid), when compared to a control reactionwithout EDTA.

In some aspects, the lipolytic enzyme variants of the disclosure have aprotease stability that is greater that the protease stability of theparent lipolytic enzyme.

In some embodiments, the lipolytic enzyme variant retains at least about40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%,about 92%, about 95%, about 96%, about 97%, about 98%, or about 99%lipolytic activity after exposure to proteolytic degradation over agiven time period, for example, at least about 10 minutes, at leastabout 20 minutes, at least about 30 minutes, at least about 40 minutes,at least at least about 60 minutes, about 90 minutes, about 120 minutes,about 160 minutes, about 180 minutes, about 210 minutes, about 240minutes, about 270 minutes, about 300 minutes, about 360 minutes, about420 minutes, about 480 minutes, about 540 minutes or about 600 minutes.

In some aspects, an improved performance (improved properties) of alipolytic enzyme variant includes a lipolytic enzyme variant withimproved wash performance and an improved detergent stability, relativeto the corresponding parent lipolytic enzyme.

In some aspects, an improved performance (improved properties) of alipolytic enzyme variant includes a lipolytic enzyme variant withimproved wash performance and an improved thermostability, relative tothe corresponding parent lipolytic enzyme.

In some aspects, an improved performance (improved properties) of alipolytic enzyme variant includes a lipolytic enzyme variant withimproved wash performance and an improved calcium-ion binding stability,relative to the corresponding parent lipolytic enzyme.

In some aspects, an improved performance (improved properties) of alipolytic enzyme variant includes a lipolytic enzyme variant withimproved wash performance and an improved protease stability, relativeto the corresponding parent lipolytic enzyme.

In some aspects, an improved performance (improved properties) of alipolytic enzyme variant includes a lipolytic enzyme variant withimproved detergent stability performance and an improvedthermostability, relative to the corresponding parent lipolytic enzyme.

In some aspects, an improved performance (improved properties) of alipolytic enzyme variant includes a lipolytic enzyme variant withimproved detergent stability performance and an improved calcium-ionbinding stability, relative to the corresponding parent lipolyticenzyme.

In some aspects, an improved performance (improved properties) of alipolytic enzyme variant includes a lipolytic enzyme variant withimproved detergent stability performance and an improved proteasestability, relative to the corresponding parent lipolytic enzyme.

In some aspects, an improved performance (improved properties) of alipolytic enzyme variant includes a lipolytic enzyme variant withimproved thermostability performance and an improved calcium-ion bindingstability, relative to the corresponding parent lipolytic enzyme.

In some aspects, an improved performance (improved properties) of alipolytic enzyme variant includes a lipolytic enzyme variant withimproved thermostability performance and an improved protease stability,relative to the corresponding parent lipolytic enzyme.

In some aspects, improved properties of a lipolytic enzyme variantincludes a lipolytic enzyme variant with improved wash performance,improved thermostability and improved calcium ion binding stability,optionally with retained or improved stability and or proteasestability, relative to the corresponding parent lipolytic enzyme.

In one embodiment, the disclosure provides a lipolytic enzyme variant oran active fragment thereof comprising at least two amino acidmodifications to a parent lipolytic enzyme, wherein the at least twoamino acid modifications are at a position of the lipolytic enzymevariant selected from the group consisting of 4, 11, 19, 22, 26, 39, 53,60, 64, 67, 70, 71, 93, 95, 111, 117, 119, 120, 121, 125, 127, 131, 134,141, 142, 143, 146, 149, 154, 159, 163, 166, 170, 179, 185, 196, 199,203, 204, 205, 206, 208, 209, 212, 217, 218, 221, 224, 227, 229, 235,247, 252, 261, 262, 265, 272, 280 and 286, wherein the amino acidpositions of the variant are numbered by correspondence with the aminoacid sequence of SEQ ID NO:1, wherein the lipolytic enzyme variant hasat least 60% sequence identity to SEQ ID NO:1. In one aspect, thelipolytic enzyme variant is derived from a parent lipolytic enzymehaving 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identityto the amino acid sequence of SEQ ID NO: 1. In one aspect, the variantcomprises an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the disclosure provides a lipolytic enzyme variant oran active fragment thereof comprising at least two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen ormore amino acid modification to a parent lipolytic enzyme, wherein theat least one amino acid modification is at a position of the lipolyticenzyme variant selected from the group consisting 4, 11, 19, 22, 26, 39,53, 60, 64, 67, 70, 71, 93, 95, 111, 117, 119, 120, 121, 125, 127, 131,134, 141, 142, 143, 146, 149, 154, 159, 163, 166, 170, 179, 185, 196,199, 203, 204, 205, 206, 208, 209, 212, 217, 218, 221, 224, 227, 229,235, 247, 252, 261, 262, 265, 272, 280 and 286, wherein the amino acidpositions of the variant are numbered by correspondence with the aminoacid sequence of SEQ ID NO:1, wherein the lipolytic enzyme variant hasat least 60% sequence identity to SEQ ID NO: 1.

Polypeptides of the Disclosure

The present disclosure provides novel polypeptides, which may becollectively referred to as “polypeptides of the disclosure.”Polypeptides of the disclosure include isolated, recombinant,substantially pure, or non-naturally occurring lipolytic enzyme variantpolypeptides, including for example, variant lipolytic enzymepolypeptides, having lipolytic activity and an improved property(performance) such as but not limiting to improved cleaning performance(such as improved wash performance), an increased expression, anincreased detergent stability, an increased thermostability, or any onecombination thereof.

In some embodiments, polypeptides of the disclosure are useful incleaning applications and can be incorporated into cleaning compositionsthat are useful in methods of cleaning an item or a surface (e.g., asurface of an item) in need of cleaning.

In some embodiments, the disclosure includes an isolated, recombinant,substantially pure, or non-naturally occurring variant polypeptidehaving lipolytic activity, which polypeptide comprises a polypeptidesequence having at least having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the variant polypeptide is a variant having aspecified degree of amino acid sequence homology to the parent lipolyticenzyme, e.g., at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or even at least 99% sequence homology to the amino acidsequence of SEQ ID NO: 1. Homology can be determined by amino acidsequence alignment, e.g., using a program such as BLAST, ALIGN, orCLUSTAL, as described herein.

In some embodiments, the variant polypeptide disclosed herein comprisesan amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acidsequence identity to the amino acid sequence of SEQ ID NO: 1.

Also provided is an isolated, recombinant, substantially pure, ornon-naturally occurring sequence which encodes a lipolytic enzymevariant having lipolytic activity, said lipolytic enzyme variant (e.g.,variant lipase) comprising an amino acid sequence which differs from theamino acid sequence of the parent lipase of SEQ ID NO:1 by no more than50, no more than 40, no more than 30, no more than 35, no more than 25,no more than 20, no more than 19, no more than 18, no more than 17, nomore than 16, no more than 15, no more than 14, no more than 13, no morethan 12, no more than 11, no more than 10, no more than 9, no more than8, no more than 7, no more than 6, no more than 5, no more than 4, nomore than 3, no more than 2, or no more than 1 amino acid residue(s),wherein amino acid positions of the variant lipase are numberedaccording to the numbering of corresponding amino acid positions in theamino acid sequence of the parent lipolytic enzyme of SEQ ID NO: 1 asdetermined by alignment of the lipolytic enzyme variant amino acidsequence with the parent lipolytic amino acid sequence of SEQ ID NO: 1.

As noted above, the lipolytic enzyme variant polypeptides of thedisclosure have enzymatic activities (e.g., lipolytic activities) andthus are useful in cleaning applications, including but not limited to,methods for cleaning dishware items, tableware items, fabrics, and itemshaving hard surfaces (e.g., the hard surface of a table, table top,wall, furniture item, floor, ceiling, medical instrument, examinationtable, etc.). Exemplary cleaning compositions comprising one or morelipolytic enzyme variant polypeptides of the disclosure are describedherein. The enzymatic activity (e.g., lipolytic enzyme activity) of alipolytic enzyme variant polypeptide of the disclosure can be determinedreadily using procedures well known to those of ordinary skill in theart.

A polypeptide of the disclosure can be subject to various changes, suchas one or more amino acid insertions, deletions, and/or substitutions,either conservative or non-conservative, including where such changes donot substantially alter the enzymatic activity of the polypeptide.Similarly, a nucleic acid of the disclosure can also be subject tovarious changes, such as one or more substitutions of one or morenucleic acids in one or more codons such that a particular codon encodesthe same or a different amino acid, resulting in either a silentvariation (e.g., mutation in a nucleotide sequence results in a silentmutation in the amino acid sequence, for example when the encoded aminoacid is not altered by the nucleic acid mutation) or non-silentvariation, one or more deletions of one or more nucleic acids (orcodons) in the sequence, one or more additions or insertions of one ormore nucleic acids (or codons) in the sequence, and/or cleavage of orone or more truncations of one or more nucleic acids (or codons) in thesequence. Many such changes in the nucleic acid sequence may notsubstantially alter the enzymatic activity of the resulting encodedlipolytic enzyme variant compared to the lipolytic enzyme variantencoded by the original nucleic acid sequence. A nucleic acid of thedisclosure can also be modified to include one or more codons thatprovide for optimum expression in an expression system (e.g., bacterialexpression system), while, if desired, said one or more codons stillencode the same amino acid(s).

In some embodiments, the present disclosure provides a genus ofpolypeptides comprising lipolytic enzyme variant polypeptides having thedesired activity or performance (e.g., lipolytic enzyme activity orcleaning performance) which comprise sequences having the amino acidsubstitutions described herein and also which comprise one or moreadditional amino acid substitutions, such as conservative andnon-conservative substitutions, wherein the polypeptide exhibits,maintains, or approximately maintains the desired enzymatic activity(e.g., lipolytic enzyme activity or lipase activity, as reflected in thecleaning performance of the lipolytic enzyme variant). Amino acidsubstitutions in accordance with the disclosure may include, but are notlimited to, one or more non-conservative substitutions and/or one ormore conservative amino acid substitutions. A conservative amino acidresidue substitution typically involves exchanging a member within onefunctional class of amino acid residues for a residue that belongs tothe same functional class (identical amino acid residues are consideredfunctionally homologous or conserved in calculating percent functionalhomology). A conservative amino acid substitution typically involves thesubstitution of an amino acid in an amino acid sequence with afunctionally similar amino acid. For example, alanine, glycine, serine,and threonine are functionally similar and thus may serve asconservative amino acid substitutions for one another. Aspartic acid andglutamic acid may serve as conservative substitutions for one another.Asparagine and glutamine may serve as conservative substitutions for oneanother. Arginine, lysine, and histidine may serve as conservativesubstitutions for one another. Isoleucine, leucine, methionine, andvaline may serve as conservative substitutions for one another.Phenylalanine, tyrosine, and tryptophan may serve as conservativesubstitutions for one another.

Other conservative amino acid substitution groups can be envisioned. Forexample, amino acids can be grouped by similar function or chemicalstructure or composition (e.g., acidic, basic, aliphatic, aromatic,sulfur-containing). For instance, an aliphatic grouping may comprise:Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I). Othergroups containing amino acids that are considered conservativesubstitutions for one another include: aromatic: Phenylalanine (F),Tyrosine (Y), Tryptophan (W); sulfur-containing: Methionine (M),Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic:Aspartic acid (D), Glutamic acid (E); non-polar uncharged residues,Cysteine (C), Methionine (M), and Proline (P); hydrophilic unchargedresidues: Serine (S), Threonine (T), Asparagine (N), and Glutamine (Q).Additional groupings of amino acids are well-known to those of skill inthe art and described in various standard textbooks. Listing of apolypeptide sequence herein, in conjunction with the above substitutiongroups, provides an express listing of all conservatively substitutedpolypeptide sequences.

More conservative substitutions exist within the amino acid residueclasses described above, which also or alternatively can be suitable.Conservation groups for substitutions that are more conservativeinclude: valine-leucine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, and asparagine-glutamine. Thus, forexample, in some embodiments, the disclosure provides an isolated orrecombinant lipolytic enzyme variant polypeptide (e.g., variant lipase)having lipolytic activity, said lipolytic enzyme variant polypeptidecomprising an amino acid sequence having at least about 90%, about 95%,about 96%, about 97%, about 98%, about 99%, or about 99.5% sequenceidentity to the amino acid sequence of SEQ ID NO:1. A conservativesubstitution of one amino acid for another in a lipolytic enzyme variantof the disclosure is not expected to alter significantly the enzymaticactivity or cleaning performance activity of the lipolytic enzymevariant. Enzymatic activity or cleaning performance activity of theresultant lipolytic enzyme can be readily determined using the standardassays and the assays described herein.

Conservatively substituted variations of a polypeptide sequence of thedisclosure (e.g., lipolytic enzyme variants of the disclosure) includesubstitutions of a small percentage, sometimes less than about 25%,about 20%, about 15%, about 14%, about 13%, about 12%, about 11%, about10%, about 9%, about 8%, about 7%, or about 6% of the amino acids of thepolypeptide sequence, or less than about 5%, about 4%, about 3%, about2%, or about 1%, of the amino acids of the polypeptide sequence, with aconservatively selected amino acid of the same conservative substitutiongroup.

Nucleic Acids of the Disclosure

The disclosure provides isolated, non-naturally occurring, orrecombinant nucleic acids (also referred to herein as“polynucleotides”), which may be collectively referred to as “nucleicacids of the disclosure” or “polynucleotides of the disclosure”, whichencode polypeptides of the disclosure. Nucleic acids of the disclosure,including all described below, are useful in recombinant production(e.g., expression) of polypeptides of the disclosure, typically throughexpression of a plasmid expression vector comprising a sequence encodingthe polypeptide of interest or fragment thereof, or through chromosomalintegration. In some aspects, a polynucleotide sequence encoding thelipolytic enzyme variant described herein (as well as other sequencesincluded in the vector) is integrated into the genome of the host cell,while in other embodiments, a plasmid vector comprising a polynucleotidesequence encoding the lipolytic enzyme variant remains as autonomousextra-chromosomal element within the cell. As discussed above,polypeptides include isolated, recombinant, substantially pure, ornon-naturally occurring lipolytic enzyme variant polypeptides, includingfor example, variant lipolytic enzyme polypeptides, having lipolyticactivity and an improved property (performance) such as but not limitingto improved cleaning performance (such as improved wash performance), anincreased expression, a decreased (reduced) malodor, an increaseddetergent stability, an increased thermostability, an increased calciumion binding stability, an increased protease stability, or any onecombination thereof, that are useful in cleaning applications andcleaning compositions for cleaning an item or a surface (e.g., surfaceof an item) in need of cleaning.

In one aspect, the polynucleotide of the disclosure is a DNA sequence ofSEQ ID NO:3 encoding the lipolytic enzyme of SEQ ID NO: 1.

In some embodiments, the disclosure provides an isolated, recombinant,substantially pure, codon optimized, or non-naturally occurring nucleicacid comprising a nucleotide sequence encoding any polypeptide(including any fusion protein, etc.) of the disclosure described herein.The disclosure also provides an isolated, recombinant, substantiallypure, or non-naturally-occurring nucleic acid comprising a nucleotidesequence encoding a combination of two or more of any polypeptides ofthe disclosure described above and elsewhere herein.

Also provided is an isolated, recombinant, substantially pure, ornon-naturally occurring nucleic acid comprising a polynucleotidesequence which encodes a lipolytic enzyme variant having lipolyticactivity, said lipolytic enzyme variant (e.g., variant lipase)comprising an amino acid sequence which differs from the amino acidsequence of SEQ ID NO:1 by no more than 50, no more than 40, no morethan 30, no more than 35, no more than 25, no more than 20, no more than19, no more than 18, no more than 17, no more than 16, no more than 15,no more than 14, no more than 13, no more than 12, no more than 11, nomore than 10, no more than 9, no more than 8, no more than 7, no morethan 6, no more than 5, no more than 4, no more than 3, no more than 2,or no more than 1 amino acid residue(s), wherein amino acid positions ofthe variant lipase are numbered according to the numbering ofcorresponding amino acid positions in the amino acid sequence of thelipolytic enzyme shown in SEQ ID NO:1 as determined by alignment of thelipolytic enzyme variant amino acid sequence with the parent lipolyticamino acid sequence of SEQ ID NO:1.

The present disclosure provides nucleic acids encoding a lipase variantof the parent lipase of SEQ ID NO:1, as described previously, whereinthe amino acid positions of the lipase variant are numbered bycorrespondence with the parent lipolytic amino acid sequence of SEQ IDNO:1. The nucleic acids encoding the parent lipase or the lipasevariants disclosed herein can be codon optimized for production in ahost cell such as, but not limiting to Escherichia coli and Bacillussubtilus.

Nucleic acids of the disclosure can be generated by using any suitablesynthesis, manipulation, and/or isolation techniques, or combinationsthereof. For example, a polynucleotide of the disclosure may be producedusing standard nucleic acid synthesis techniques, such as solid-phasesynthesis techniques that are well-known to those skilled in the art.The synthesis of the nucleic acids of the disclosure can be alsofacilitated (or alternatively accomplished) by any suitable method knownin the art, including but not limited to chemical synthesis using theclassical phosphoramidite method (See e.g., Beaucage et al. TetrahedronLetters 22:1859-69 (1981)); or the method described by Matthes et al.(See, Matthes et al., EMBO J. 3:801-805 (1984), as is typicallypracticed in automated synthetic methods. Nucleic acids of thedisclosure also can be produced by using an automatic DNA synthesizer.Customized nucleic acids can be ordered from a variety of commercialsources (e.g., The Midland Certified Reagent Company, the Great AmericanGene Company, Operon Technologies Inc., and DNA2.0). Other techniquesfor synthesizing nucleic acids and related principles are known in theart (See e.g., Itakura et al., Ann. Rev. Biochem. 53:323 (1984); andItakura et al., Science 198:1056 (1984)).

Methods for Making Modified Lipolytic Enzyme Variants of the Disclosure

A variety of methods are known in the art that are suitable forgenerating modified polynucleotides of the disclosure that encodelipolytic enzyme variants of the disclosure, including, but not limitedto, for example, site-saturation mutagenesis, scanning mutagenesis,insertional mutagenesis, deletion mutagenesis, random mutagenesis,site-directed mutagenesis, and directed-evolution, as well as variousother recombinatorial approaches. Methods for making modifiedpolynucleotides and proteins (e.g., lipolytic enzyme variants) includeDNA shuffling methodologies, methods based on non-homologousrecombination of genes, such as ITCHY (See, Ostermeier et al., 7:2139-44(1999)), SCRACHY (See, Lutz et al. 98:11248-53 (2001)), SHIPREC (See,Sieber et al., 19:456-60 (2001)), and NRR (See, Bittker et al.,20:1024-9 (2001); Bittker et al., 101:7011-6 (2004)), and methods thatrely on the use of oligonucleotides to insert random and targetedmutations, deletions and/or insertions (See, Ness et al., 20:1251-5(2002); Coco et al., 20:1246-50 (2002); Zha et al., 4:34-9 (2003);Glaser et al., 149:3903-13 (1992)).

Vectors, Cells, and Methods for Producing Lipolytic Enzyme Variants ofthe Disclosure

The present disclosure provides isolated or recombinant vectorscomprising at least one polynucleotide of the disclosure describedherein (e.g., a polynucleotide encoding a lipolytic enzyme variant ofthe disclosure described herein), isolated or recombinant expressionvectors or expression cassettes comprising at least one nucleic acid orpolynucleotide of the disclosure, isolated, substantially pure, orrecombinant DNA constructs comprising at least one nucleic acid orpolynucleotide of the disclosure, isolated or recombinant cellscomprising at least one polynucleotide of the disclosure, cell culturescomprising cells comprising at least one polynucleotide of thedisclosure, cell cultures comprising at least one nucleic acid orpolynucleotide of the disclosure, and compositions comprising one ormore such vectors, nucleic acids, expression vectors, expressioncassettes, DNA constructs, cells, cell cultures, or any combination ormixtures thereof.

In some embodiments, the disclosure provides recombinant cellscomprising at least one vector (e.g., expression vector or DNAconstruct) of the disclosure which comprises at least one nucleic acidor polynucleotide of the disclosure. Some such recombinant cells aretransformed or transfected with such at least one vector. Such cells aretypically referred to as host cells. Some such cells comprise bacterialcells, including, but are not limited to Bacillus sp. cells, such asBacillus subtilis cells. The disclosure also provides recombinant cells(e.g., recombinant host cells) comprising at least one lipolytic enzymevariant of the disclosure.

In some aspects, the lipolytic variants described herein are encoded bya DNA sequence that is codon optimized for expression in Escherichiacoli. In some aspects, the lipolytic variants described herein areencoded by a DNA sequence that is codon optimized for expression inBacillus subtilus.

In some embodiments, the disclosure provides a vector comprising anucleic acid or polynucleotide of the disclosure. In some embodiments,the vector is an expression vector or expression cassette in which apolynucleotide sequence of the disclosure which encodes a lipolyticenzyme variant of the disclosure is operably linked to one or additionalnucleic acid segments required for efficient gene expression (e.g., apromoter operably linked to the polynucleotide of the disclosure whichencodes a lipolytic enzyme variant of the disclosure). A vector mayinclude a transcription terminator and/or a selection gene, such as anantibiotic resistance gene that enables continuous cultural maintenanceof plasmid-infected host cells by growth in antimicrobial-containingmedia.

An expression vector may be derived from plasmid or viral DNA, or inalternative embodiments, contains elements of both. Exemplary vectorsinclude, but are not limited to pXX, pC194, pJH101, pE194, pHP13 (See,Harwood and Cutting [eds.], Chapter 3, Molecular Biological Methods forBacillus, John Wiley & Sons [1990]; suitable replicating plasmids for B.subtilis include those listed on p. 92; See also, Perego, IntegrationalVectors for Genetic Manipulations in Bacillus subtilis, in Sonenshein etal., [eds.] Bacillus subtilis and Other Gram-Positive Bacteria:Biochemistry, Physiology and Molecular Genetics, American Society forMicrobiology, Washington, D.C. [1993], pp. 615-624.

For expression and production of a protein of interest (e.g., lipolyticenzyme variant) in a cell, at least one expression vector comprising atleast one copy of a polynucleotide encoding the modified lipolyticenzyme, and preferably comprising multiple copies, is transformed intothe cell under conditions suitable for expression of the lipolyticenzyme. In some embodiments of the present disclosure, a polynucleotidesequence encoding the lipolytic enzyme variant described herein (as wellas other sequences included in the vector) is integrated into the genomeof the host cell, while in other embodiments, a plasmid vectorcomprising a polynucleotide sequence encoding the lipolytic enzymevariant remains as autonomous extra-chromosomal element within the cell.The disclosure provides both extrachromosomal nucleic acid elements aswell as incoming nucleotide sequences that are integrated into the hostcell genome. The vectors described herein are useful for production ofthe lipolytic enzyme variants of the disclosure. In some embodiments, apolynucleotide construct encoding the lipolytic enzyme variant ispresent on an integrating vector that enables the integration andoptionally the amplification of the polynucleotide encoding thelipolytic enzyme variant into the bacterial chromosome. Examples ofsites for integration are well known to those skilled in the art. Insome embodiments, transcription of a polynucleotide encoding a lipolyticenzyme variant of the disclosure is effectuated by a promoter that isthe wild-type promoter for the selected precursor lipolytic enzyme. Insome other embodiments, the promoter is heterologous to the precursorlipolytic enzyme, but is functional in the host cell. Specifically,examples of suitable promoters for use in microorganisms include, butare not limited to these described in WO2017/152169 (herein incorporatedby reference) as well as promoters for use in bacterial host cellsinclude, but are not limited to, for example, the amyE, amyQ, amyL,pstS, sacB, pSPAC, pAprE, pVeg, pHpaII promoters, the promoter of the B.stearothermophilus maltogenic amylase gene, the B. amyloliquefaciens(BAN) amylase gene, the B. subtilis alkaline lipolytic enzyme gene, theB. clausii alkaline lipolytic enzyme gene the B. pumilis xylosidasegene, the B. thuringiensis cryIIIA, and the B. lichenformisalpha-amylase gene. Additional promoters include, but are not limited tothe A4 promoter, as well as phage Lambda P_(R) or P_(L) promoters, andthe E. coli lac, trp or tac promoters.

Lipolytic enzyme variants of the present disclosure can be produced inany suitable host cells including but not limiting to suitableGram-positive microorganism, bacteria and fungi. For example, in someembodiments, the lipolytic enzyme variant is produced in host cells offungal and/or bacterial origin. In some embodiments, the host cells areBacillus sp., Streptomyces sp., Escherichia sp. or Aspergillus sp. Insome embodiments, the lipolytic enzyme variants are produced by Bacillussp. host cells. Examples of Bacillus sp. host cells that find use in theproduction of the lipolytic enzyme variants of the disclosure include,but are not limited to B. lichenformis, B. lentus, B. subtilis, B.lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. coagulans,B. circulans, B. pumilis, B. thuringiensis, B. clausii, and B.megaterium, as well as other organisms within the genus Bacillus. Insome embodiments, B. subtilis host cells are used for production oflipolytic enzyme variants. U.S. Pat. Nos. 5,264,366 and 4,760,025 (RE34,606) describe various Bacillus host strains that can be used forproducing lipolytic enzyme variants of the disclosure, although othersuitable strains can be used.

Several industrial bacterial strains that can be used to producelipolytic enzyme variants of the disclosure include non-recombinant(i.e., wild-type) Bacillus sp. strains, as well as variants ofnaturally-occurring strains and/or recombinant strains. In someembodiments, the host strain is a recombinant strain, wherein apolynucleotide encoding a polypeptide of interest has been introducedinto the host. In some embodiments, the host strain is a B. subtilishost strain and particularly a recombinant Bacillus subtilis hoststrain. Numerous B. subtilis strains are known, including, but notlimited to for example, 1A6 (ATCC 39085), 168 (1A01), SB19, W23, Ts85,B637, PB1753 through PB1758, PB3360, JH642, 1A243 (ATCC 39,087), ATCC21332, ATCC 6051, MI113, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP211 strain (See e.g., Hoch et al., Genetics 73:215-228 [1973]; See also,U.S. Pat. Nos. 4,450,235 and 4,302,544, and EP 0134048, each of which isincorporated by reference in its entirety). The use of B. subtilis as anexpression host cells is well known in the art (See e.g., Palva et al.,Gene 19:81-87 [1982]; Fahnestock and Fischer, J. Bacteriol., 165:796-804[1986]; and Wang et al., Gene 69:39-47 [1988]).

In some embodiments, the Bacillus host cell is a Bacillus sp. thatincludes a mutation or deletion in at least one of the following genes,degU, degS, degR and degQ. Preferably the mutation is in a degU gene,and more preferably the mutation is degU(Hy)32 (See e.g., Msadek et al.,J. Bacteriol. 172:824-834 [1990]; and Olmos et al., Mol. Gen. Genet.253:562-567 [1997]). One suitable host strain is a Bacillus subtiliscarrying a degU32(Hy) mutation. In some embodiments, the Bacillus hostcomprises a mutation or deletion in scoC4 (See e.g., Caldwell et al., J.Bacteriol. 183:7329-7340 [2001]); spoIIE (See e.g., Arigoni et al., Mol.Microbiol. 31:1407-1415 [1999]); and/or oppA or other genes of the oppoperon (See e.g., Perego et al., Mol. Microbiol. 5:173-185 [1991]).Indeed, it is contemplated that any mutation in the opp operon thatcauses the same phenotype as a mutation in the oppA gene will find usein some embodiments of the altered Bacillus strain of the disclosure. Insome embodiments, these mutations occur alone, while in otherembodiments, combinations of mutations are present. In some embodiments,an altered Bacillus host cell strain that can be used to produce alipolytic enzyme variant of the disclosure is a Bacillus host strainthat already includes a mutation in one or more of the above-mentionedgenes. In addition, Bacillus sp. host cells that comprise mutation(s)and/or deletions of endogenous lipolytic enzyme genes find use. In someembodiments, the Bacillus host cell comprises a deletion of the aprE andthe nprE genes. In other embodiments, the Bacillus sp. host cellcomprises a deletion of 5 lipolytic enzyme genes, while in otherembodiments, the Bacillus sp. host cell comprises a deletion of 9lipolytic enzyme genes (See e.g., U.S. Pat. Appln. Pub. No.2005/0202535, incorporated herein by reference).

Host cells are transformed with at least one nucleic acid encoding atleast one lipolytic enzyme variant of the disclosure using any suitablemethod known in the art. Whether the nucleic acid is incorporated into avector or is used without the presence of plasmid DNA, it is typicallyintroduced into a microorganism, in some embodiments, preferably an E.coli cell or a competent Bacillus cell. Methods for introducing anucleic acid (e.g., DNA) into Bacillus cells or E. coli cells utilizingplasmid DNA constructs or vectors and transforming such plasmid DNAconstructs or vectors into such cells are well known. In someembodiments, the plasmids are subsequently isolated from E. coli cellsand transformed into Bacillus cells. However, it is not essential to useintervening microorganisms such as E. coli, and in some embodiments, aDNA construct or vector is directly introduced into a Bacillus host.

Those of skill in the art are well aware of suitable methods forintroducing nucleic acid or polynucleotide sequences of the disclosureinto Bacillus cells (See e.g., Ferrari et al., “Genetics,” in Harwood etal. [eds.], Bacillus, Plenum Publishing Corp. [1989], pp. 57-72;Saunders et al., J. Bacteriol. 157:718-726 [1984]; Hoch et al., J.Bacteriol. 93:1925-1937 [1967]; Mann et al., Current Microbiol.13:131-135 [1986]; Holubova, Folia Microbiol. 30:97 [1985]; Chang etal., Mol. Gen. Genet. 168:11-115 [1979]; Vorobjeva et al., FEMSMicrobiol. Lett. 7:261-263 [1980]; Smith et al., Appl. Env. Microbiol.51:634 [1986]; Fisher et al., Arch. Microbiol. 139:213-217 [1981]; andMcDonald, J. Gen. Microbiol. 130:203 [1984]). Indeed, such methods astransformation, including protoplast transformation and congression,transduction, and protoplast fusion are well known and suited for use inthe present disclosure. Methods of transformation are used to introducea DNA construct or vector comprising a nucleic acid encoding a lipolyticenzyme variant of the present disclosure into a host cell. Methods knownin the art to transform Bacillus cells include such methods as plasmidmarker rescue transformation, which involves the uptake of a donorplasmid by competent cells carrying a partially homologous residentplasmid (See, Contente et al., Plasmid 2:555-571 [1979]; Haima et al.,Mol. Gen. Genet. 223:185-191 [1990]; Weinrauch et al., J. Bacteriol.154:1077-1087 [1983]; and Weinrauch et al., J. Bacteriol. 169:1205-1211[1987]). In this method, the incoming donor plasmid recombines with thehomologous region of the resident “helper” plasmid in a process thatmimics chromosomal transformation.

In addition to commonly used methods, in some embodiments, host cellsare directly transformed with a DNA construct or vector comprising anucleic acid encoding a lipolytic enzyme variant of the disclosure(i.e., an intermediate cell is not used to amplify, or otherwiseprocess, the DNA construct or vector prior to introduction into the hostcell). Introduction of the DNA construct or vector of the disclosureinto the host cell includes those physical and chemical methods known inthe art to introduce a nucleic acid sequence (e.g., DNA sequence) into ahost cell without insertion into a plasmid or vector. Such methodsinclude, but are not limited to calcium chloride precipitation,electroporation, naked DNA, liposomes and the like. In additionalembodiments, DNA constructs or vector are co-transformed with a plasmid,without being inserted into the plasmid. In further embodiments, aselective marker is deleted from the altered Bacillus strain by methodsknown in the art (See, Stahl et al., J. Bacteriol. 158:411-418 [1984];and Palmeros et al., Gene 247:255-264 [2000]).

In some embodiments, the transformed cells of the present disclosure arecultured in conventional nutrient media. The suitable specific cultureconditions, such as temperature, pH and the like are known to thoseskilled in the art and are well described in the scientific literature.In some embodiments, the disclosure provides a culture (e.g., cellculture) comprising at least one lipolytic enzyme variant or at leastone nucleic acid of the disclosure. Also provided are compositionscomprising at least one nucleic acid, vector, or DNA construct of thedisclosure.

In some embodiments, host cells transformed with at least onepolynucleotide sequence encoding at least one lipolytic enzyme variantof the disclosure are cultured in a suitable nutrient medium underconditions permitting the expression of the present lipolytic enzyme,after which the resulting lipolytic enzyme is recovered from theculture. The medium used to culture the cells comprises any conventionalmedium suitable for growing the host cells, such as minimal or complexmedia containing appropriate supplements. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedrecipes (See e.g., the catalogues of the American Type CultureCollection). In some embodiments, the lipolytic enzyme produced by thecells is recovered from the culture medium by conventional procedures,including, but not limited to for example, separating the host cellsfrom the medium by centrifugation or filtration, precipitating theproteinaceous components of the supernatant or filtrate by means of asalt (e.g., ammonium sulfate), chromatographic purification (e.g., ionexchange, gel filtration, affinity, etc.). Any method suitable forrecovering or purifying a lipolytic enzyme variant finds use in thepresent disclosure.

In some embodiments, a lipolytic enzyme variant produced by arecombinant host cell is secreted into the culture medium. A nucleicacid sequence that encodes a purification facilitating domain may beused to facilitate purification of soluble proteins. A vector or DNAconstruct comprising a polynucleotide sequence encoding a lipolyticenzyme variant may further comprise a nucleic acid sequence encoding apurification facilitating domain to facilitate purification of thelipolytic enzyme variant (See e.g., Kroll et al., DNA Cell Biol.12:441-53 [1993]). Such purification facilitating domains include, butare not limited to, for example, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals (See, Porath, Protein Expr. Purif. 3:263-281 [1992]), protein Adomain that allow purification on immobilized immunoglobulin, and thedomain utilized in the FLAGS extension/affinity purification system(e.g., protein A domains available from Immunex Corp., Seattle, Wash.).The inclusion of a cleavable linker sequence such as Factor XA orenterokinase (e.g., sequences available from Invitrogen, San Diego,Calif.) between the purification domain and the heterologous proteinalso find use to facilitate purification.

Assays for detecting and measuring the enzymatic activity of an enzyme,such as a lipolytic enzyme variant of the disclosure, are well known.Various assays for detecting and measuring activity of lipolytic enzymes(e.g., lipolytic enzyme variants of the disclosure), are also known tothose of ordinary skill in the art. A variety of methods can be used todetermine the level of production of a mature lipolytic enzyme (e.g.,mature lipolytic enzyme variants of the present disclosure) in a hostcell. Such methods include, but are not limited to, methods that utilizeeither polyclonal or monoclonal antibodies specific for the lipolyticenzyme. Exemplary methods include, but are not limited to enzyme-linkedimmunosorbent assays (ELISA), radioimmunoassays (RIA), fluorescentimmunoassays (FIA), and fluorescent activated cell sorting (FACS). Theseand other assays are well known in the art (See e.g., Maddox et al., J.Exp. Med. 158:1211 [1983]).

In some other embodiments, the disclosure provides methods for making orproducing a mature lipolytic enzyme variant of the disclosure. A maturelipolytic enzyme variant does not include a signal peptide or apropeptide sequence. Some methods comprise making or producing alipolytic enzyme variant of the disclosure in a recombinant bacterialhost cell, such as for example, a Bacillus sp. cell (e.g., a B. subtiliscell). In some embodiments, the disclosure provides a method ofproducing a lipolytic enzyme variant of the disclosure, the methodcomprising cultivating a recombinant host cell comprising a recombinantexpression vector comprising a nucleic acid encoding a lipolytic enzymevariant of the disclosure under conditions conducive to the productionof the lipolytic enzyme variant. Some such methods further compriserecovering the lipolytic enzyme variant from the culture.

In some embodiments the disclosure provides methods of producing alipolytic enzyme variant of the disclosure, the methods comprising: (a)introducing a recombinant expression vector comprising a nucleic acidencoding a lipolytic enzyme variant of the disclosure into a populationof cells (e.g., bacterial cells, such as B. subtilis cells); and (b)culturing the cells in a culture medium under conditions conducive toproduce the lipolytic enzyme variant encoded by the expression vector.Some such methods further comprise: (c) isolating the lipolytic enzymevariant from the cells or from the culture medium.

Compositions Comprising Lipolytic Enzyme Variants of the Disclosure

Some further embodiments are directed to compositions comprising one ormore lipolytic enzyme variants described herein, such as but not limitedto cleaning compositions.

Cleaning compositions and cleaning formulations include any compositionthat is suited for cleaning, bleaching, disinfecting, and/or sterilizingany object, item, and/or surface. Such compositions and formulationsinclude, but are not limited to for example, liquid and/or solidcompositions, including cleaning or detergent compositions (e.g.,liquid, tablet, gel, bar, granule, pouch, and/or solid laundry cleaningor detergent compositions and fine fabric detergent compositions; hardsurface (such as but not limiting to the hard surface of a table, tabletop, wall, furniture item, floor, ceiling, medical instrument,examination table, etc.) cleaning compositions and formulations, such asfor glass, wood, ceramic and metal counter tops and windows; carpetcleaners; oven cleaners; fabric fresheners; fabric softeners; andtextile, laundry booster cleaning or detergent compositions, laundryadditive cleaning compositions, and laundry pre-spotter cleaningcompositions; dishwashing compositions, including hand or manualdishwash compositions (e.g., “hand” or “manual” dishwashing detergents)and automatic dishwashing compositions (e.g., “automatic dishwashingdetergents”).

Cleaning composition or cleaning formulations, as used herein, include,unless otherwise indicated, granular or powder-form all-purpose orheavy-duty washing agents, especially cleaning detergents; liquid,granular, gel, solid, tablet, or paste-form all-purpose washing agents,especially the so-called heavy-duty liquid (HDL) detergent or heavy-dutypowder detergent (HDD) types; liquid fine-fabric detergents; hand ormanual dishwashing agents, including those of the high-foaming type;hand or manual dishwashing, automatic dishwashing, or dishware ortableware washing agents, including the various tablet, powder, solid,granular, liquid, gel, and rinse-aid types for household andinstitutional use; liquid cleaning and disinfecting agents, includingantibacterial hand-wash types, cleaning bars, mouthwashes, denturecleaners, car shampoos, carpet shampoos, bathroom cleaners; hairshampoos and/or hair-rinses for humans and other animals; shower gelsand foam baths and metal cleaners; as well as cleaning auxiliaries, suchas bleach additives and “stain-stick” or pre-treat types. In someembodiments, granular compositions are in “compact” form; in someembodiments, liquid compositions are in a “concentrated” form.

In some embodiments, the cleaning compositions of the present disclosureare provided in unit dose form, including tablets, capsules, sachets,pouches, and multi-compartment pouches. In some embodiments, the unitdose format is designed to provide controlled release of the ingredientswithin a multi-compartment pouch (or other unit dose format). Suitableunit dose and controlled release formats are known in the art (See e.g.,EP 2 100 949, WO 02/102955, U.S. Pat. Nos. 4,765,916 and 4,972,017, andWO 04/111178 for materials suitable for use in unit dose and controlledrelease formats). In some embodiments, the unit dose form is provided bytablets wrapped with a water-soluble film or water-soluble pouches.Various formats for unit doses are provided in EP 2 100 947, and areknown in the art.

In some embodiments of the present disclosure, the cleaning compositionscomprise at least one lipolytic enzyme variant of the present disclosureat a level from about 0.00001% to about 10% of enzyme protein by weightof the composition and the balance (e.g., about 99.999% to about 90.0%)comprising cleaning adjunct materials by weight of composition. In someembodiments of the present disclosure, the cleaning compositions of thepresent disclosure comprises at least one lipolytic enzyme variant at alevel of about 0.00001% to about 10%, about 0.00001% to about 5%, about0.0001% to 1%, about 0.001% to 0.5%, about 0.001% to about 2%, about0.005% to about 0.5%, about 0.01% to 0.2% of enzyme protein by weight ofthe composition and the balance of the cleaning composition (e.g., about99.9999% to about 90.0%, about 99.999% to about 98%, about 99.995% toabout 99.5% by weight) comprising cleaning adjunct materials.

In some embodiments, the cleaning composition is a granular or powderlaundry detergent. In some embodiments, the cleaning composition is aliquid laundry detergent or a dish washing detergent.

As used herein, the term “detergent composition” or “detergentformulation” is used in reference to a composition intended for use in awash medium for the cleaning of soiled or dirty objects, includingparticular fabric and/or non-fabric objects or items. Such compositionsof the present disclosure are not limited to any particular detergentcomposition or formulation. Indeed, in some embodiments, the detergentsof the disclosure comprise at least one lipolytic enzyme variant of thedisclosure and, in addition, one or more surfactants, transferase(s),hydrolytic enzymes, oxido reductases, builders (e.g., a builder salt),bleaching agents, bleach activators, bluing agents, fluorescent dyes,caking inhibitors, masking agents, enzyme activators, antioxidants,and/or solubilizers. In some instances, a builder salt is a mixture of asilicate salt and a phosphate salt, preferably with more silicate (e.g.,sodium metasilicate) than phosphate (e.g., sodium tripolyphosphate).Some compositions of the disclosure, such as, but not limited to,cleaning compositions or detergent compositions, do not contain anyphosphate (e.g., phosphate salt or phosphate builder).

Unless otherwise noted, all component or composition levels providedherein are made in reference to the active level of that component orcomposition, and are exclusive of impurities, for example, residualsolvents or by-products, which may be present in commercially availablesources. Enzyme components weights are based on total active protein.All percentages and ratios are calculated by weight unless otherwiseindicated. All percentages and ratios are calculated based on the totalcomposition unless otherwise indicated. In the exemplified detergentcompositions, the enzymes levels are expressed by pure enzyme by weightof the total composition and unless otherwise specified, the detergentingredients are expressed by weight of the total compositions.

In some embodiments, the lipolytic enzyme variants of the presentdisclosure can be used in compositions comprising an adjunct materialand a lipolytic enzyme variant, wherein the composition is cleaningcomposition.

In some embodiments, the cleaning compositions of the present disclosurecomprises one or more lipolytic enzymes and adjunct materials. In someembodiments, these adjuncts are incorporated for example, to assist orenhance cleaning performance, for treatment of the substrate to becleaned, or to modify the aesthetics of the cleaning composition as isthe case with perfumes, colorants, dyes or the like. It is understoodthat such adjuncts are in addition to the lipolytic enzyme variants ofthe present disclosure. The precise nature of these additionalcomponents, and levels of incorporation thereof, will depend on thephysical form of the composition and the nature of the cleaningoperation for which it is to be used. Suitable adjunct materialsinclude, but are not limited to, surfactants (for example, surfactantsthat are efficient in removal of fatty acids from the fabric), builders,chelating agents, dye transfer inhibiting agents, dye transfer agents,deposition aids, dispersants, additional enzymes, and enzymestabilizers, catalytic materials, bleaching agents, bleach activators,bleach catalysts, bleach boosters, hydrogen peroxide, sources ofhydrogen peroxide, preformed peracids, polymeric dispersing agents, claysoil removal/anti-redeposition agents, brighteners, optical brighteners,suds suppressors, dyes, perfumes, structure elasticizing agents, fabricconditioners, fabric softeners, carriers, hydrotropes, processing aidsand/or pigments, preservatives, anti-oxidants, anti-shrinkage agents,anti-wrinkle agents, germicides, fungicides, filler salts, hydrotropes,photoactivators, fluorescers, colorants, color speckles, silvercare,anti-tarnish and/or anti-corrosion agents, alkalinity sources,solubilizing agents, carriers, processing aids, pigments, soil releasepolymers, dispersants, other enzymes, enzyme stabilizing systems and pHcontrol agents. In addition to the disclosure below, suitable examplesof such other adjuncts and levels of use are found in U.S. Pat. Nos.5,576,282, 6,306,812, and 6,326,348, incorporated by reference. Theaforementioned adjunct ingredients may constitute the balance of thecleaning compositions of the present disclosure.

In embodiments in which the cleaning adjunct materials are notcompatible with the lipolytic enzyme variants of the present disclosurein the cleaning compositions, then suitable methods of keeping thecleaning adjunct materials and the lipolytic enzyme(s) separated (i.e.,not in contact with each other) until combination of the two componentsis appropriate are used. Such separation methods include any suitablemethod known in the art (e.g., gelcaps, encapsulation, tablets, physicalseparation, etc.).

In some embodiments, the adjuvant and lipolytic enzyme are present in asingle composition. In other embodiments, the adjuvant and lipolyticenzyme are present in separate compositions that are combined beforecontacting an oil stain on fabric, or combined on the oil stain.

The present cleaning compositions can include one or more adjuvants foruse in combination with a lypolytic enzyme. Suitable adjuvants can havea relatively small hydrophilic portion with no net charge andhydrophobic portion that is linear or saturated. In some embodiments,the hydrophobic portion includes at least, six, seven, eight, or nineadjacent aliphatic carbons. In some embodiments, the hydrophobic portionis cyclic. In some embodiments, the hydrophobic portion is not branched.Suitable adjuvants include surfactancts including sugar-based compoundsand zwitterionic compounds. Suitable adjuvants are disclosed, and herebyincorporated by reference in its entirety, in WO2011078949.

Sugar-based surfactants include maltopyranosides, thiomaltopyransodies,glucopyranosides, and their derivatives. Maltose-based surfactants weregenerally more effective than glucose-based surfactants. In someembodiments, a preferred sugar-based surfactant has a hydrophobic tailchain length of at least 4, at least 5, at least 6, and even at least 7carbons. The tail can be aliphatic or cyclic. The tail can beunbranched, although branching is acceptable with sufficient chainlength.

Particular examples of sugar-based surfactants includenonyl-β-D-maltopyranoside, decyl-β-D-maltopyranoside,undecyl-β-D-maltopyranoside, dodecyl-β-D-maltopyranoside,tridecyl-β-D-maltopyranoside, tetradecyl-β-D-maltopyranoside,hexaecyl-β-D-maltopyranoside, n-dodecyl-β-D-maltopyranoside and thelike, 2,6-dimethyl-4-heptyl-β-D-maltopyranoside,2-propyl-1-pentyl-β-D-maltopyranoside, nonyl-β-D-glucopyranoside,nonyl-β-D-glucopyranoside, decyl-β-D-glucopyranoside,dodecyl-β-D-glucopyranoside, sucrose monododecanoate, certaincyclohexylalkyl-β-D-maltosides (e.g., the CYMAL®s and CYGLAs), and theMEGA™ surfactants.

The adjuvant can be a non-sugar, non-ionic surfactant. Exemplarysurfactants include alkyl ethoxylates and alkylphenol ethoxylates,Tritons with an ethoxylate repeat of nine or less. Particular Tritonsare ANAPOE®-X-100 and ANAPOE®-X-114. In some embodiments, the adjuvantis a non-ionic phosphine oxide surfactant, having a hydrophobic tail ofat least about 9 carbons. Exemplary surfactants includedimethyldecylphoshine oxide and dimethyldodecylphoshine oxide.

The adjuvant can be a zwitterionic surfactant, such as a FOS-choline. Insome embodiments, the FOS-choline has a hydrophobic tail with a chainlength of 12 or greater. The hydrophobic tail can be saturated andunsaturated and can be cyclic. Exemplary FOS-choline surfactants includeFOS-CHOLINE®-12, FOS-CHOLINE®-13, FOS-CHOLINE®-14, LYSOFOS-CHOLINE®-14,FOS-CHOLINE®-15, FOS-CHOLINE®-16, FOS-MEA®-12, DODECAFOS, ISO unsat11-10, ISO 11-6, CYOFO, NOPOL-FOS, CYCLOFOS® (CYMAL®)-5, -6. -7, -8,etc., and the like.

In some cases, the adjuvant can be a sulfobetaine zwitterionicsurfactant. Preferred sulfobetaine surfactants have a hydrophobic tailhaving at least 12 carbons, e.g., ANZERGENT® 3-12 and ANZERGENT® 3-14.The zwitterionic oxides and CHAPS-based surfactants (e.g. CHAPS andCHAPSO) are also effective, typically at higher doses than thesulfobetaines.

In some cases, the adjuvant can be an anionic detergent, for example, asarcosine. Preferred sarcosines have a hydrophobic tail having at least10 carbons. In some cases, the adjuvant can also be deoxycholate.

In some embodiments, the cleaning compositions comprising at least onelipolytic enzyme variant comprise one or more of the followingingredients (based on total composition weight): from about 0.0005 wt %to about 0.5 wt %, from about 0.001 wt % to about 0.1 wt %, or even fromabout 0.002 wt % to about 0.05 wt % of said lipolytic enzyme variant;and one or more of the following: from about 0.00003 wt % to about 0.1wt % fabric hueing agent; from about 0.001 wt % to about 5 wt %, perfumecapsules; from about 0.001 wt % to about 1 wt %, cold-water solublebrighteners; from about 0.00003 wt % to about 0.1 wt % bleach catalysts;from about 0.00003 wt % to about 0.1 wt % bacterial cleaning cellulases;and/or from about 0.05 wt % to about 20 wt % Guerbet nonionicsurfactants.

The adjuvant can be present in a composition in an amount of at least0.001%, at least 0.005%, at least 0.01%, at least 0.05%, at least 0.1%,or more, or at least 0.01 ppm, at least 0.05 ppm, at least 0.1 ppm, atleast 0.5 ppm, at least 1 ppm, at least 5 ppm, at least 10 ppm, or more.In some cases, the adjuvant may be present in a preselected range, e.g.,about 0.001-0.01%, about 0.01-0.1%, about 0.1-1%, or about 0.01-1 ppm,about 0.1-1 ppm, or about 1-10 ppm. In some cases, optimum activity isobserved over a range, above and below which activity is reduced.

The surfactant system of the detergent can comprise nonionic, anionic,cationic, ampholytic, and/or zwitterionic surfactants. The surfactant istypically present at a level from 0.1% to 60% by weight, e.g. 1% to 40%,particularly 10-40% preferably from about 3% to about 20% by weight. Thedetergent will usually contain 0-50% of anionic surfactant such aslinear alkylbenzenesulfonate (LAS), alpha-olefin sulfonate (AOS), alkylsulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS orAES), secondary alkane sulfonates (SAS), alpha-sulfo fatty acid methylesters, alkyl- or alkenylsuccinic acid or soap.

The detergent can comprise 0-40% of nonionic surfactant polyalkyleneoxide (e.g. polyethylene oxide) condensates of alkyl phenols. Preferrednonionic surfactants are alcohol ethoxylate (AEO or AE), carboxylatedalcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside,alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fattyacid monoethanolamide, alkyl(N-methyl)-glucoseamide or polyhydroxy alkylfatty acid amide (e.g. as described in WO 92106154).

Semi-polar nonionic surfactants are another category of nonionicsurfactants which include water-soluble amine oxides containing onealkyl moiety of from about 10 to about 18 carbon atoms and 2 moietiesselected from the group consisting of alkyl groups and hydroxyalkylgroups containing from about 1 to about 3 carbon atoms; water solublephosphine oxides containing one alkyl moiety of from about 10 to about18 carbon atoms and 2 moieties selected from the group consisting ofalkyl groups and hydroxyalkyl groups containing from about 1 to about 3carbon atoms; and water-soluble sulfoxides containing one alkyl moietyfrom about 10 no to about 18 carbon atoms and a moiety selected from thegroup consisting of alkyl and hydroxyalkyl moieties of from about 1 toabout 3 carbon atoms. The amine oxide surfactants in particular includeC₁₀-C₁₈ alkyl dimethyl amine oxides and C₈-C₁₂ alkoxy ethyl dihydroxyethyl amine oxides.

The detergent composition can further comprise cationic surfactants.Cationic detersive surfactants used are those having one long-chainhydrocarbyl group. Examples of such cationic surfactants include theammonium surfactants such as alkyl trimethyl ammonium halogenides.Highly preferred cationic surfactants are the water soluble quaternaryammonium compounds. Examples of suitable quaternary ammonium compoundsinclude coconut trimethyl ammonium chloride or bromide; coconut methyldihydroxy ethyl ammonium chloride or bromide; decyl triethyl ammoniumchloride; decyl dimethyl hydroxyl ethyl ammonium chloride or bromide;C12-15 dimethyl hydroxyl ethyl ammonium chloride or bromide; coconutdimethyl hydroxyl ethyl ammonium chloride or bromide; myristyl trimethylammonium methyl sulphate; lauryl dimethyl benzyl ammonium chloride orbromide; lauryl dimethyl (ethenoxy)₄ ammonium chloride or bromide;choline esters, dialkyl imidazolines.

The detergent composition can further comprise ampholytic surfactants.These surfactants can be broadly described as aliphatic derivatives ofsecondary or tertiary amines, or aliphatic derivatives of heterocyclicsecondary and tertiary amines in which the aliphatic radical can bestraight-, or branched-chain. One of the aliphatic substituent containsat least about 8 carbon atoms, typically from about 8 to about 18 carbonatoms, and at least one contains an anionic water-solubilizing group,e.g. carboxy, sulfonate, sulfate. Examples of compounds falling withinthis definition are sodium 3-(dodecylamino) propionate, sodium3-(dodecylamino)-propane-1-sulfonate, sodium 2-(dodecylamino)ethylsulfate, sodium 2-(dimethylamino)octadecanoate, di-sodium3-(N-carboxymethyldodecylamino)propane-I-sulfonate, disodiumoctadecyl-iminodiacetate, sodium 1-carboxymethyl-2-undecylimidazole, andsodium N,N-bis(2-hydroxyethyl)-2-sulfato-3-dodecoxy-propylamine. Sodium3-(dodecylamino )propane-I-sulfonate is preferred.

Zwitterionic surfactants are also used in detergent compositionsespecially within laundry. These surfactants can be broadly described asderivatives of secondary and tertiary amines, derivatives ofheterocyclic secondary and tertiary amines, or derivatives of quaternaryammonium, quaternary phosphonium or tertiary sulfonium compounds. Thecationic atom in the quaternary compound can be part of a heterocyclicring. In all of these compounds, there is at least one aliphatic group,straight chain or branched, containing from about 3 to 18 carbon atomsand at least one aliphatic substituent containing an anionic watersolubilizing group, e.g. carboxy, sulfonate, sulfate, phosphate orphosphonate. Ethoxylated zwitterionic compounds in combination withzwitterionic surfactants have been particularly used for clay soilremoval in laundry applications.

In some embodiments in which the cleaning compositions of the presentdisclosure are formulated as compositions suitable for use in laundrymachine washing method(s), the compositions of the present disclosurepreferably contain at least one surfactant and at least one buildercompound, as well as one or more cleaning adjunct materials preferablyselected from organic polymeric compounds, bleaching agents, additionalenzymes, suds suppressors, dispersants, lime-soap dispersants, soilsuspension and anti-redeposition agents and corrosion inhibitors. Insome embodiments, laundry compositions also contain softening agents(i.e., as additional cleaning adjunct materials).

The detergent may contain 1-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate, citrate,nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).The detergent may also be unbuilt i.e. essentially free of detergentbuilder.

The detergent builders may be subdivided into phosphorus-containing andnon-phosphorous-containing types. Examples of phosphorus-containinginorganic alkaline detergent builders include the water-soluble salts,especially alkali metal pyrophosphates, orthophosphates, polyphosphatesand phosphonates. Examples of non-phosphorus-containing inorganicbuilders include water soluble alkali metal carbonates, borates andsilicates as well as layered disilicates and the various types of waterinsoluble crystalline or amorphous alumino silicates of which zeolitesis the best known representative. Examples of suitable organic buildersinclude alkali metal, ammonium or substituted ammonium salts ofsuccinates, malo nates, fatty acid malonates, fatty acid sulphonates,carboxymethoxy succinates, poly acetates, carboxylates,polycarboxylates, aminopolycarboxylates and polyacetyl carboxylates.

A suitable chelant for inclusion in the detergent compositions isethylenediamine-N,N′-disuccinic acid (EDDS) or the alkali metal,alkaline earth metal, ammonium, or substituted ammonium salts thereof,or mixtures thereof. Some EDDS compounds are the free acid form and thesodium or magnesium salt thereof. Examples of such sodium salts of EDDSinclude Na2EDDS and Na4EDDS. Examples of such magnesium salts of EDDSinclude MgEDDS and Mg2EDDS.

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose (CMC), poly (vinylpyrrolidone) (PVP),polyethyleneglycol (PEG), poly (vinyl alcohol) (PVA), polycarboxylatessuch as polyacrylates, maleic/acrylic acid copolymers and laurylmethacrylate/acrylic acid copolymers.

The detergent composition may contain bleaching agents of thechlorine/bromine-type or the oxygen-type. The bleaching agents may becoated or encapsulated. Examples of inorganic chlorine/bromine-typebleaches are lithium, sodium or calcium hypochlorite or hypobromite aswell as chlorinated trisodium phosphate. The bleaching system may alsocomprise a hydrogen peroxide source such as perborate or percarbonatewhich may be combined with a peracid-forming bleach activator such astetraacetyl-ethylenediamine (TAED) or nonanoyloxybenzenesulfonate(NOBS). Examples of organic chlorine/bromine-type bleaches areheterocyclic N-bromo and N-chloro imides such as trichloroisocyanuric,tribromoisocyanuric, dibromoisocyanuric and dichloroisocyanuric acids,and salts thereof with water solubilizing cations such as potassium andsodium. Hydantoin compounds are also suitable. The bleaching system mayalso comprise peroxyacids of, e.g., the amide, imide, or sulfone type.

In dishwashing detergents, the oxygen bleaches are preferred, forexample in the form of an inorganic persalt, preferably with a bleachprecursor or as a peroxy acid compound. Typical examples of suitableperoxy bleach compounds are alkali metal perborates, both tetrahydratesand monohydrates, alkali metal percarbonates, persilicates andperphosphates. Preferred activator materials aretetraacetylethylenediamine (TAED), nonanoyloxybenzenesulfonate (NOBS),3,5-trimethyl-hexsanoloxybenzenesulfonate (ISONOBS) orpentaacetylglucose (PAG).

In some embodiments, bleaches, bleach activators and/or bleach catalystsare present in the compositions of the present disclosure. In someembodiments, the cleaning compositions of the present disclosurecomprise inorganic and/or organic bleaching compound(s). Inorganicbleaches include, but are not limited to perhydrate salts (e.g.,perborate, percarbonate, perphosphate, persulfate, and persilicatesalts). In some embodiments, inorganic perhydrate salts are alkali metalsalts. In some embodiments, inorganic perhydrate salts are included asthe crystalline solid, without additional protection, although in someother embodiments, the salt is coated. Any suitable salt known in theart finds use in the present disclosure (See e.g., EP 2 100 949).

In some embodiments, bleach activators are used in the compositions ofthe present disclosure. Bleach activators are typically organic peracidprecursors that enhance the bleaching action in the course of cleaningat temperatures of 60° C. and below. Bleach activators suitable for useherein include compounds which, under perhydrolysis conditions, givealiphatic peroxoycarboxylic acids having preferably from about 1 toabout 10 carbon atoms, in particular from about 2 to about 4 carbonatoms, and/or optionally substituted perbenzoic acid. Additional bleachactivators are known in the art and find use in the present disclosure(See e.g., EP 2 100 949).

In addition, in some embodiments and as further described herein, thecleaning compositions of the present disclosure further comprise atleast one bleach catalyst. In some embodiments, the manganesetriazacyclononane and related complexes find use, as well as cobalt,copper, manganese, and iron complexes. Additional bleach catalysts finduse in the present disclosure (See e.g., U.S. Pat. Nos. 4,246,612,5,227,084, 4,810,410, WO 99/06521, and EP 2 100 949).

In some still further embodiments, the cleaning compositions providedherein contain at least one deposition aid. Suitable deposition aidsinclude, but are not limited to, polyethylene glycol, polypropyleneglycol, polycarboxylate, soil release polymers such as polytelephthalicacid, clays such as kaolinite, montmorillonite, atapulgite, illite,bentonite, halloysite, and mixtures thereof.

As indicated herein, in some embodiments, anti-redeposition agents finduse in some embodiments of the present disclosure. In some embodiments,non-ionic surfactants find use. For example, in automatic dishwashingembodiments, non-ionic surfactants find use for surface modificationpurposes, in particular for sheeting, to avoid filming and spotting andto improve shine. These non-ionic surfactants also find use inpreventing the re-deposition of soils. In some embodiments, theanti-redeposition agent is a non-ionic surfactant as known in the art(See e.g., EP 2 100 949).

In some embodiments, the cleaning compositions of the present disclosureinclude one or more dye transfer inhibiting agents. Suitable polymericdye transfer inhibiting agents include, but are not limited to,polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers ofN-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones andpolyvinylimidazoles or mixtures thereof. In embodiments in which atleast one dye transfer inhibiting agent is used, the cleaningcompositions of the present disclosure comprise from about 0.0001% toabout 10%, from about 0.01% to about 5%, or even from about 0.1% toabout 3% by weight of the cleaning composition.

In some embodiments, silicates are included within the compositions ofthe present disclosure. In some such embodiments, sodium silicates(e.g., sodium disilicate, sodium metasilicate, and crystallinephyllosilicates) find use. In some embodiments, silicates are present ata level of from about 1% to about 20%. In some embodiments, silicatesare present at a level of from about 5% to about 15% by weight of thecomposition.

In some still additional embodiments, the cleaning compositions of thepresent disclosure also contain dispersants. Suitable water-solubleorganic materials include, but are not limited to the homo- orco-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms.

In one aspect, the amount of lipolytic enzyme variant protein of thedisclosure may be 0.001-30 mg per gram of detergent or 0.001-100 mg perliter of wash liquor. The lipase variants of the disclosure areparticularly suited for detergents comprising of a combination ofanionic and nonionic surfactant with 70-100% by weight of anionicsurfactant and 0-30% by weight of nonionic, particularly 80-100% ofanionic surfactant, and 0-20% nonionic surfactant. As further described,some preferred lipases of the disclosure are also suited for detergentscomprising 40-70% anionic and 30-60% non-ionic surfactant.

In some embodiments, the cleaning compositions of the present disclosurecontain one or more catalytic metal complexes. In some embodiments, ametal-containing bleach catalyst finds use. In some embodiments, themetal bleach catalyst comprises a catalyst system comprising atransition metal cation of defined bleach catalytic activity, (e.g.,copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganesecations), an auxiliary metal cation having little or no bleach catalyticactivity (e.g., zinc or aluminum cations), and a sequestrate havingdefined stability constants for the catalytic and auxiliary metalcations, particularly ethylenediaminetetraacetic acid,ethylenediaminetetra (methylenephosphonic acid) and water-soluble saltsthereof are used (See e.g., U.S. Pat. No. 4,430,243). In someembodiments, the cleaning compositions of the present disclosure arecatalyzed by means of a manganese compound. Such compounds and levels ofuse are well known in the art (See e.g., U.S. Pat. No. 5,576,282). Inadditional embodiments, cobalt bleach catalysts find use in the cleaningcompositions of the present disclosure. Various cobalt bleach catalystsare known in the art (See e.g., U.S. Pat. Nos. 5,597,936 and 5,595,967)and are readily prepared by known procedures.

In some additional embodiments, the cleaning compositions of the presentdisclosure include a transition metal complex of a macropolycyclic rigidligand (MRL). As a practical matter, and not by way of limitation, insome embodiments, the compositions and cleaning processes provided bythe present disclosure are adjusted to provide on the order of at leastone part per hundred million of the active MRL species in the aqueouswashing medium, and in some embodiments, provide from about 0.005 ppm toabout 25 ppm, more preferably from about 0.05 ppm to about 10 ppm, andmost preferably from about 0.1 ppm to about 5 ppm, of the MRL in thewash liquor.

In some embodiments, transition-metals in the instant transition-metalbleach catalyst include, but are not limited to manganese, iron andchromium. MRLs also include, but are not limited to special ultra-rigidligands that are cross-bridged (e.g.,5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane). Suitabletransition metal MRLs are readily prepared by known procedures (Seee.g., WO 2000/32601, and U.S. Pat. No. 6,225,464).

In some embodiments, the cleaning compositions of the present disclosurecomprise metal care agents. Metal care agents find use in preventingand/or reducing the tarnishing, corrosion, and/or oxidation of metals,including aluminum, stainless steel, and non-ferrous metals (e.g.,silver and copper). Suitable metal care agents include those describedin EP 2 100 949, WO 9426860 and WO 94/26859). In some embodiments, themetal care agent is a zinc salt. In some further embodiments, thecleaning compositions of the present disclosure comprise from about 0.1%to about 5% by weight of one or more metal care agents.

The detergent composition may, in addition to the lipolytic enzymevariant of the disclosure, comprise other enzyme(s) providing cleaningperformance and/or fabric care benefits, e.g. proteases, additionallipases, cutinases, amylases, cellulases, peroxidases, oxidases (e.g.laccases), mannanases, oxidoreductases, and/or pectate lyases.

In some embodiments, the cleaning compositions of the present disclosurecomprise one or more additional detergent enzymes, which providecleaning performance and/or fabric care and/or dishwashing benefits.Examples of suitable enzymes include, but are not limited to,hemicellulases, cellulases, perhydrolases, peroxidases, lipolyticenzymes, xylanases, lipases, phospholipases, esterases, cutinases,pectinases, pectate lyases, mannanases, keratinases, reductases,oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases,tannases, pentosanases, malanases, 8-glucanases, arabinosidases,hyaluronidases, chondroitinases, laccases, and amylases, or anycombinations or mixtures thereof. In some embodiments, a combination ofenzymes is used (i.e., a “cocktail”) comprising conventional applicableenzymes like lipolytic enzyme, lipase, cutinase and/or cellulase inconjunction with amylase is used.

For example, a lipolytic enzyme variant of the disclosure can becombined with a protease. Suitable proteolytic enzymes include those ofanimal, vegetable or microbial origin. In some embodiments, microbialproteolytic enzymes are used. In some embodiments, the proteolyticenzyme is preferably an alkaline microbial proteolytic enzyme or atrypsin-like proteolytic enzyme. Examples of alkaline lipolytic enzymesinclude lipases, especially those derived from Bacillus (e.g., lentus,amyloliquefaciens, Carlsberg, 309, 147 and 168). Additional examplesinclude those mutant proteolytic enzymes described in U.S. Pat. Nos. RE34,606, 5,955,340, 5,700,676, 6,312,936, and 6,482,628, all of which areincorporated herein by reference. Additional protease examples include,but are not limited to trypsin (e.g., of porcine or bovine origin), andthe Fusarium protease enzyme described in WO 89/06270. In someembodiments, commercially available protease enzymes that find use inthe present disclosure include, but are not limited to MAXATASE®,MAXACAL™, MAXAPEM™, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT®,PURAFECT® OXP, PURAMAX™ EXCELLASE™, and PURAFAST™ (Genencor); ALCALASE®,SAVINASE®, PRIMASE®, DURAZYM™, POLARZYME®, OVOZYME®, KANNASE®,LIQUANASE®, NEUTRASE®, RELASE® and ESPERASE® (Novozymes); BLAP™ andBLAP™ variants (Henkel Kommanditgesellschaft auf Aktien, Duesseldorf,Germany), and KAP (B. alkalophilus lipase; Kao Corp., Tokyo, Japan).Various proteolytic enzymes are described in WO95/23221, WO 92/21760,U.S. Pat. Publ. No. 2008/0090747, and U.S. Pat. Nos. 5,801,039,5,340,735, 5,500,364, 5,855,625, U.S. RE 34,606, 5,955,340, 5,700,676,6,312,936, and 6,482,628, and various other patents. In some furtherembodiments, metalloprotease enzymes find use in the present disclosure,including but not limited to the neutral metalloprotease enzymedescribed in WO 07/044993.

In one embodiment, the lipolytic enzyme variant or active fragmentdescribed herein has a protease stability that is greater that theprotease stability of the parent lipolytic enzyme.

In some embodiments of the present disclosure, any suitable amylasefinds use in the present disclosure. In some embodiments, any amylase(e.g., alpha and/or beta) suitable for use in alkaline solutions alsofind use. Suitable amylases include, but are not limited to those ofbacterial or fungal origin. Chemically or genetically modified mutantsare included in some embodiments. Amylases that find use in the presentdisclosure, include, but are not limited to a-amylases obtained from B.lichenformis (See e.g., GB 1,296,839). Commercially available amylasesthat find use in the present disclosure include, but are not limited toDURAMYL®, TERMAMYL®, FUNGAMYL®, STAINZYME®, STAINZYME PLUS®, STAINZYMEULTRA®, and BAN™ (Novozymes), as well as POWERASE™, RAPIDASE® andMAXAMYL® P (Genencor).

In some embodiments of the present disclosure, the cleaning compositionsof the present disclosure further comprise amylases at a level fromabout 0.00001% to about 10% of additional amylase by weight of thecomposition and the balance of cleaning adjunct materials by weight ofcomposition. In some other embodiments of the present disclosure, thecleaning compositions of the present disclosure also comprise amylasesat a level of about 0.0001% to about 10%, about 0.001% to about 5%,about 0.001% to about 2%, about 0.005% to about 0.5% amylase by weightof the composition.

In some further embodiments, any suitable cellulase finds used in thecleaning compositions of the present disclosure. Suitable cellulasesinclude, but are not limited to those of bacterial or fungal origin.Chemically or genetically modified mutants are included in someembodiments. Suitable cellulases include, but are not limited toHumicola insolens cellulases (See e.g., U.S. Pat. No. 4,435,307).Especially suitable cellulases are the cellulases having color carebenefits (See e.g., EP 0 495 257). Commercially available cellulasesthat find use in the present include, but are not limited to CELLUZYME®,CAREZYME® (Novozymes), and KAC-500(B)™ (Kao Corporation) PURADAX HA1200E (Danisco), PURADAX EG 7000L(Danisco). In some embodiments,cellulases are incorporated as portions or fragments of mature wild-typeor variant cellulases, wherein a portion of the N-terminus is deleted(See e.g., U.S. Pat. No. 5,874,276). In some embodiments, the cleaningcompositions of the present disclosure further comprise cellulases at alevel from about 0.00001% to about 10% of additional cellulase by weightof the composition and the balance of cleaning adjunct materials byweight of composition. In some other embodiments of the presentdisclosure, the cleaning compositions of the present disclosure alsocomprise cellulases at a level of about 0.0001% to about 10%, about0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5%cellulase by weight of the composition.

Any mannanase suitable for use in detergent compositions also finds usein the present disclosure. Suitable mannanases include, but are notlimited to those of bacterial or fungal origin. Chemically orgenetically modified mutants are included in some embodiments. Variousmannanases are known which find use in the present disclosure (See e.g.,U.S. Pat. Nos. 6,566,114, 6,602,842, and 6,440,991, all of which areincorporated herein by reference). In some embodiments, the cleaningcompositions of the present disclosure further comprise mannanases at alevel from about 0.00001% to about 10% of additional mannanase by weightof the composition and the balance of cleaning adjunct materials byweight of composition. In some embodiments of the present disclosure,the cleaning compositions of the present disclosure also comprisemannanases at a level of about 0.0001% to about 10%, about 0.001% toabout 5%, about 0.001% to about 2%, about 0.005% to about 0.5% mannanaseby weight of the composition.

In some embodiments, peroxidases are used in combination with hydrogenperoxide or a source thereof (e.g., a percarbonate, perborate orpersulfate) in the compositions of the present disclosure. In somealternative embodiments, oxidases are used in combination with oxygen.Both types of enzymes are used for “solution bleaching” (i.e., toprevent transfer of a textile dye from a dyed fabric to another fabricwhen the fabrics are washed together in a wash liquor), preferablytogether with an enhancing agent (See e.g., WO 94/12621 and WO95/01426). Suitable peroxidases/oxidases include, but are not limited tothose of plant, bacterial or fungal origin. Chemically or geneticallymodified mutants are included in some embodiments. In some embodiments,the cleaning compositions of the present disclosure further compriseperoxidase and/or oxidase enzymes at a level from about 0.00001% toabout 10% of additional peroxidase and/or oxidase by weight of thecomposition and the balance of cleaning adjunct materials by weight ofcomposition. In some other embodiments of the present disclosure, thecleaning compositions of the present disclosure also comprise,peroxidase and/or oxidase enzymes at a level of about 0.0001% to about10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% toabout 0.5% peroxidase and/or oxidase enzymes by weight of thecomposition.

In some embodiments, additional enzymes find use, including but notlimited to perhydrolases (See e.g., WO 05/056782). In addition, in someembodiments, mixtures of the above-mentioned enzymes are encompassedherein, in particular one or more additional lipolytic enzymes, amylase,protease, mannanase, and/or at least one cellulase. Indeed, it iscontemplated that various mixtures of these enzymes will find use in thepresent disclosure. It is also contemplated that the varying levels ofthe lipolytic enzyme variant(s) and one or more additional enzymes mayboth independently range to about 10%, the balance of the cleaningcomposition being cleaning adjunct materials. The specific selection ofcleaning adjunct materials is readily made by considering the surface,item, or fabric to be cleaned, and the desired form of the compositionfor the cleaning conditions during use (e.g., through the wash detergentuse).

The enzymes of the detergent composition may be stabilized usingconventional stabilizing agents (e.g. a polyol such as propylene glycolor glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or aboric acid derivative as e.g. an aromatic borate ester). Boronic acid orborinic acid derivatives as enzyme stabilizers include Boric acid,Thiophene-3-boronic acid, Thiophene-2-boronic acid,4-Methylthiophene-2-boronic acid, 5-Ethylthiophene-2-boronic acid,5-Methylthiophene-2-boronic acid, 5-Bromothiophene-2-boronic acid,5-Chlorothiophene-2-boronic acid, Dibenzothiophene-1-boronic acid,Dibenzofuran-1-boronic acid, Dibenzofnran-4-boronic acid,Picoline-2-boronic acid, Diphenylborinic acid (ethanolamine complex),5-Methoxythio-phene-2-boronic acid, Thionaphthrene-1-boronic acid,Furan-2-boronic acid, Furan-3-boronic acid,2,5-dimethyl-thiophene-3-boronic acid, Benzofuran-1-boronic acid,3-Methoxythio-phene-2-boronic acid, 5-n-Propyl-thiophene-2-boronic acid,5-Methoxyfuran-2-boronic acid, 3-Bromothiophene-2-boronic acid,5-Ethylfuran-2-boronic acid, 4-Carbazole ethyl boronic acid.

In some embodiments, the enzymes used in the cleaning compositions arestabilized by any suitable technique. In some embodiments, the enzymesemployed herein are stabilized by the presence of water-soluble sourcesof calcium and/or magnesium ions in the finished compositions thatprovide such ions to the enzymes. In some embodiments, the enzymestabilizers include oligosaccharides, polysaccharides, and inorganicdivalent metal salts, including alkaline earth metals, such as calciumsalts. It is contemplated that various techniques for enzymestabilization will find use in the present disclosure. For example, insome embodiments, the enzymes employed herein are stabilized by thepresence of water-soluble sources of zinc (II), calcium (II) and/ormagnesium (II) ions in the finished compositions that provide such ionsto the enzymes, as well as other metal ions (e.g., barium (II), scandium(II), iron (II), manganese (II), aluminum (III), Tin (II), cobalt (II),copper (II), nickel (II), and oxovanadium (IV). Chlorides and sulfatesalso find use in some embodiments of the present disclosure. Examples ofsuitable oligosaccharides and polysaccharides (e.g., dextrins) are knownin the art (See e.g., WO 07/145964). In some embodiments, reversibleenzyme inhibitors also find use, such as boron-containing compounds(e.g., borate, 4-formyl phenyl boronic acid) and/or a tripeptidealdehyde find use to further improve stability, as desired.

An optional ingredient in cleaning compositions of the presentdisclosure is a suds suppressor (e.g. exemplified by silicones-alkylatedpolysiloxane materials, and silica-silicone mixtures, where the silicais in the form of silica aerogels and xerogels and hydrophobic silicasof various types. The suds suppressor can be incorporated asparticulates, in which the suds suppressor is advantageously releasableincorporated in a water-soluble or water dispersible, substantially nonsurface-active detergent impermeable carrier. Alternatively, the sudssuppressor can be dissolved or dispersed in a liquid carrier and appliedby spraying on to one or more of the other components.

The detergent may also contain inorganic or organic softening agents.Inorganic softening agents are exemplified by the smectite clays (5% to15%). Organic fabric softening agents (0.5% to 5%) include the waterinsoluble tertiary amines and their combination with mono C₁₂-C₁₄quaternary ammonium salts and di-long-chain amides, or high molecularweight polyethylene oxide materials.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, deflocculantmaterial, foam boosters/foam depressors (in dishwashing detergents foamdepressors), anti-corrosion agents, soil-suspending or dispersing agents(0 to 10%), anti-soil-redeposition agents, dyes, dehydrating agents,bactericides, optical brighteners, abrasives, tarnish inhibitors,coloring agents, and/or encapsulated or non-encapsulated perfumes.

Some examples of detergent formulations are shown in Tables 1-11.

TABLE 1 Liquid detergent formulation Nonionic (Neodol 25-7) AE 25% Anionic (Vista C-S50) LAS 5% Triethanolamine 5% Ethanol 10%  Stabilizer0.5, 2.5, 5% Protease 1% Amylase   0.3% Water up to 100% Adjust to pH =9.0 Lipase insert after amylase 0.001-1%

TABLE 2 Detergent formulations I, II, II, IV, V and VI. Detergentformulations I II III IV V VI (%) (%) (%) (%) (%) (%) pow- pow- pow-pow- liq- liq- Ingredients der der der der uid uid Linear  7-12  6-115-9  8-12 15-21 15-21 alkylbenzenesulfonate (calculated as acid) orslkyl sulfate, alpha olefin sulfonate, alpha-sulfo fatty acid methylesters, alkanesulfonates, soap Alcohol ethoxysulfate 1-4 1-3 — (e.g.C₁₂₋₁₈ alcohol 1-2 EO) or alkyl sulfate (e.g. C₁₆₋₁₈) soap as fatty acid— — 1-3  3-13  3-10 (e.g. C₁₆₋₂₂ or oleic acid) Alcohol ethoxylate (e.g.5-9 5-9  7-14 10-25 12-18 3-9 C₁₄₋₁₅ or C₁₂₋₁₅ 7EO or 5EO)Alkenylsuccinic  0-13 acid (C₁₂₋₁₄) Aminoethanol  8-18 sodium carbonate14-20 15-21 10-17 14-22 (Na₂CO₃) soluble silicate 2-6 1-4 3-9 1-5 (asNa₂O, 2SiO₂ ) zeolite (as NaAlSiO₄) 15-22 24-34 23-33 25-35 14-22 sodiumsulfate 0-6 4-10 0-4  0-10 (as Na₂SO₄) sodium citrate/citric acid  0-15 0-15 2-8 9-18 (C₆H₅Na₃O/C₆H₈O₇) or potassium citrate sodium perborate(as 11-18 —  8-16 0-2 0-2 NaBO₃•H₂O) or borate (as B₄O₇) TAED 2-6 — 2-8Phosphonate (e.g. — — 0-1 0-3 EDTMPA) Ethanol 0-3 carboxymethy1cellulose0-2 0-2 0-2 0-2 0-2 Polymers (PEG, PVP) 0-3 0-3 Anchoring polymers 0-31-6 1-3 1-3 0-3 (e.g. maleic/acrylic acid copolymer PVP, PEG) Propyleneglycol  8-14 Glycerol 0-5 Enzymes (alkaline 0-5 0-5 0-5 0-5 0-5 0-5lipases) minor ingredients (e.g. 0-5 0-5 0-5 0-5 0-5 0-5 suds,supressors, perfume, optical brightener, photobleach)

TABLE 3 Detergent formulations VII, VIII, IX, X, XI and XII Detergentformulations VII VIII IX X XI XII (%) (%) (%) (%) (%) (%) pow- pow- pow-liq- liq- pow- Ingredients der der der uid uid der Linear  8-14  6-1215-23 20-32 25-40 alkylbenzenesulfonate (calculated as acid) or slkylsulfate, alpha olefin sulfonate, alpha-sulfo fatty acid methyl esters,alkanesulfonates, soap Fatty alcohol sulfate  5-10 Ethoxylated fattyacid 3-9  5-11 monoethanol amide Alcohol ethoxysulfate  8-15 (e.g.C₁₂₋₁₈ alcohol 1-2 EO, or C₁₂₋₁₅ 2-3 EO) or alkyl sulfate (e.g. C₁₆₋₁₈)soap as fatty acid 0-3 0-3 2-6 0-3 (e.g. C₁₆₋₂₂ or oleic acid or lauricacid) Alcohol ethoxylate (e.g. 1-4 3-9  6-12  1-10 C₁₄₋₁₅ or C₁₂₋₁₅ 7EOor 5EO) Alkenylsuccinic acid (C₁₂₋₁₄) Aminoethanol 1-5 2-6 sodiumcarbonate  5-10  4-10 14-22  8-25 (Na₂CO₃) soluble silicate 1-4 1-4 5-15 (as Na₂O, 2SiO₂ ) zeolite (as NaAlSiO₄) 20-40 30-50 18-32 15-28sodium sulfate 2-8  3-11  5-20 0-5 (as Na₂SO₄) sodium citrate/ 5-12 3-8 5-10  8-14 citric acid (C₆H₅Na₃O/C₆H₈O₇) or potassium citrateHydrotrope (eg sodium 2-6 toluenesulfonate) sodium perborate (as 12-184-9 0-2 1-3  0-20 NaBO₃•H₂O, or NaBO₃•4H₂O) or borate (as B₄O₇) TAED (orNOBS) 2-7 1-5 0-5 Phosphonate (e.g. EDTMPA) Ethanol 1-3carboxymethylcellulose 0-2 0-1 Polymers (PEG, PVP) Anchoring 1-5 1-5 1-50-3 polymers (e.g. maleic/acrylic acid copolymer PVP, PEG) Propyleneglycol 2-5 Glycerol 3-8 Enzymes (alkaline 0-5 0-5 0-5 0-5 0-5 0-5lipases) minor ingredients 0-5 0-5 0-5 0-5 0-5 0-3 (e.g. suds,supressors, perfume, optical brightener, photobleach)

TABLE 4 Detergent composition Ex1, Ex2, Ex3 and Ex4 Detergentcomposition Material Ex. 1 Ex. 2 Ex. 3 Ex. 4 Level Level Level Level(parts (parts (parts (parts Ingredients as is) as is) as is) as is)Glycerol 3.17 3.17 3.17 3.17 MPG 5.7 5.7 5.7 5.7 NaOH 2.13 2.13 2.132.13 TEA 2.05 2.05 2.05 2.05 Neodol 25-7 12.74 12.74 12.74 12.74 F-Dye0.18 0.18 0.18 0.18 Citric Acid 1.71 1.71 1.71 1.71 LAS (as LAS 8.498.49 8.49 8.49 Acid) Fatty acid 3.03 3.03 3.03 3.03 Empigen BB 1.5 1.51.5 1.5 SLES 4.24 4.24 4.24 4.24 Dequest 2066 0.875 0.875 0.875 0.875Patent Blue 0.00036 0.00036 0.00036 0.00036 Acid Yellow 0.00005 0.000050.00005 0.00005 Opacifier 0.0512 0.0512 0.0512 0.0512 Perfume 0.7340.734 0.734 0.734 Borax 10 10 10 10 Savinase 2.362 2.362 2.362 2.362Stainzyme 0.945 0.945 0.945 0.945 Soap 3.03 3.03 3.03 3.03 EPEI 20E0 5.55.5 5.5 9 (ex Nippon Shokubai) polyethyl- eneimine having a weightaverage molecular weight of about 600, and wherein the polyethyl-eneimine has been modified by alkoxylation with an average 20 ethyleneoxide moieties LIPASE 3 3 3 3 Texcare 0 7.5 0 0 SRN170 (ex Clariant)soil release polymer Sokolan CP5 0 0 20 0 (ex BASF) Soil-release polymer

TABLE 5 Enzymatic detergent and bleaching composition A Enzymaticdetergent and bleaching composition Ingredients % by weight Sodiumdodecyl 6.5 benzene sulphonate C14-C15 primary 2 alcohol, condensed with11 moles of ethylene oxide Sodium stearate 1 Sodium silicate 7 Sodiumcarboxymethyl 0.5 cellulose Na₂SO₄ 37 Pentasodium triphosphate 15Trisodium orthophosphate 5 Fluorescer 0.2 Ethylene diamine 0.5tetraacetic acid Water 6.2 Dyes 0.01 Lipase 0.001-1 bleach systemssodium perborate + SNOBS, sodium perborate + TAED, DPDA, MPS Allgenerating 1.5 mmol peracid in solution

TABLE 6 Enzymatic detergent and bleaching composition B Enzymaticdetergent and bleaching composition Ingredients % by weight Sodiumdodecyl benzene sulphonate 8.5 C14-C15 primary alcohol, condensed 4 with11 moles of ethylene oxide sodium hardened rapeseed oil soap 1.5 sodiumtriphosphate 33 sodium carbonate 5 sodium silicate 6 sodium sulphate 20water 9 fluorescers, soil-suspending agents, minor amount dyes, perfumesanti-foam granules 1.2 Dequest R 2047 (34% pure) 0.3 Lipase 0.001-1

TABLE 7 Enzymatic Detergent compositions Detergent compositionsIngredients % wt % wt sodium alkylbenzenesulphonate 24 28 pentasodiumtripolyphosphate 15 2.1 alkaline sodium silicate sodium 10 12carboxymethylcellulose sodium 0.6 0.6 sulphate 32.5 15.4 fluorescer 0.40.4 sodium carbonate 10 35 miscellaneous + water to 100% to 100% Lipase0.001-1 0.001-1

TABLE 8 Enzymatic Detergent compositions Enzymatic Detergent compositionIngredients % by weight sodium linear dodecylbenzenesulphonate 13.35sodium C12-C13 alcohol (6.5 E0) sulphate 6.67 sodium carbonate 54.2sodium tripolyphosphate 9.01. sodium silicate 4.6 sodium hydroxide 1.66sodium carboxymethylcellulose 0.5 Dequest 2006 1.9 perfume, dye, waterq.s. Lipase 0.001-1 Protease (Alcalase) 20 GU/mL

TABLE 9 Liquid laundry detergent formulation Liquid laundry detergentformulation Ingredients Parts by weight Sodium dodecyl benzenesulphonate 8.5 C12-C15 primary alcohol, condensed 4 with 7 moles ofethylene oxide Sodium-hardened rapeseed oil soap 1.5 Sodium triphosphate33 Sodium carbonate 5 Sodium silicate 6 Sodium sulphate 20 Water 9Fluorescers, soil-suspending agents, minor amount dyes, perfumes Sodiumperborate 12 Tetraacetyl ethylene diamine (TAED) (granules) 2Proteolytic enzyme (Savinase ex NOVO) 0.4 Lipase 0.001-1 Protease(Alcalase) 20 GU/mL

TABLE 10 Liquid detergent compositions A, B, C and D Liquid detergentcompositions A B C D sodium dodecylbenzene sulphonate 9 9 9 9 C13-C15linear primary alcohol, 1 4 4 1 condensed with 7 moles of ethylene oxide(e.g. Synperonic A7) C13-C15 linear primary alcohol, 3 0 0 3 condensedwith 3 moles of ethylene oxide (e.g. Synperonic A3) sodiumtripolyphosphate 23 23 0 0 zeolite type 4A 0 0 24 24 copolymer ofacrylic acid 0 0 4 4 with maleic anhydride sodium polyacrylate 2 2 0 0alkaline silicate 5 5 fluorescer 0.25 0.25 0.16 0.16 EDTA 0.15 0.15 0.180.18 SCMC 0.5 0.5 0.55 0.55 salt 2 2 0 0 sodium sulphate 26.8 26.8 22.3122.31 sodium carbonate 0 0 10.3 10.3 moisture 10 10 11 11 TAED 3 3 3.33.3 sodium perborate monohydrate 10 10 8 8 calcium Dequest ®²⁰⁴⁷ 0.7 0.70.3 0.3 foam depressor 3 3 2.5 2.5 perfume 0.2 0.2 0 0 alkaline protease(Savinase (A) 6T) 0.4 0.4 0.4 0.4 Lipase 0.001-1

TABLE 11 Dishwashing composition Dishwashing composition Ingredients %by weight sodium tripolyphosphate 24 soda ash 20 sodium disilicate 11linear C10 alcohol, condensed with 6 moles of 2.5 ethylene oxide and 24moles of propylene oxide sodium sulphate 44 water to 100 Lipase 0.001-1

In one aspect the deterrent is an Anionic Model Detergent A, comprisinga model granular detergent (90% anionic out of total surfactants, pH insolution 10.2) made by mixing the following ingredients (% byweight):8.7% anionic surfactant: LAS (C₁₀-C₁₃), 7.4% anionic surfactant:AS (C₁₂), 1.8% Nonionic surfactant: alcohol ethoxylate (C₁₂-C₁₅ 7EO),30% Zeolite P (Wessalite P), 18% Sodium Carbonate, 500 Sodium Citrate,17% Sodium sulfate, 0.3% Carboxy-Methyl-Cellulose, 6.5%Sodium-percarbonate monohydrate, and 2.1% NOBS.

In one aspect the deterrent is an Anionic Model Detergent B, comprisinga second model granular detergent (7900 anionic out of totalsurfactants, pH in solution 10.2) made by mixing the followingingredients (% by weight): 27% anionic surfactant: AS (C₁₂), 7% Nonionicsurfactant (C₁₂₋₁₅, 7EO), 60% Zeolite P (Wessalite P), 500 SodiumCarbonate, 0.6% Sokalan CP5, 1.5% Carboxy-Methyl-Cellulose.

In one aspect the deterrent is an Anionic/non-ionic Model Detergent C,comprising a model detergent solution (32% anionic out of totalsurfactant, pH 10.2) that is made by adding the following ingredients to3.2 mM Ca2+/Mg2+(5:1) in pure water: 0.300 g/l of alkyl sulphate (AS;C₁₄₋₁₆); 0.650 g/l of alcohol ethoxylate (AEO; C₁₂₋₁₄, 6EO); 1.750 g/lof Zeolite P, 0.145 g/l of Na₂CO₃, 0.020 g/l of Sokalan CP5 and 0.050g/l of CMC (carboxy-methyl cellulose)

In one aspect the detergent is a European laundry powder detergentcomprising the following ingredients: 15% of surfactant of which 6% wasLAS, 3% was AES and 6% was non ionic surfactants, and further contained47% builder comprising fatty acid, zeolite A, carbonate and silicate; or15% of surfactant of which 3% was AES, 6% was LAS and 6% was non ionicsurfactants, and further comprised 47% builder comprising fatty acid,zeolite A, carbonate, silicate, and it comprised 5% polycarboxylatepolymers; or 15% of surfactant of which 3% was AES, 6% was LAS and 6%was non ionic surfactants, and further contained 47% builder comprisingfatty acid, zeolite A, carbonate, silicate, and it comprised 5%polycarboxylate polymers; or 15% of surfactant of which 6% was LAS, 3%was AES and 6% was nonionic surfactants and further contained 47%builder consisting of fatty acid, zeolite A, carbonate & silicate, 5%polycarboxylate dispersing polymers, 15% sodium perborate, and 4%tetraacetyl-ethylene-diamine (TAEO); or 15% of surfactant of which 6%was LAS, 3% was AES and 6% was non ionic surfactants and furthercontained 47% builder consisting of fatty acid, 22% zeolite A, carbonateand silicate, and 5% polycarboxylate dispersing polymers; or 15% ofsurfactant of which 6% was LAS, 3% was AES and 6% was non ionicsurfactants and further contained 47% builder consisting of fatty acid,22% zeolite A, carbonate and silicate, and 5% polycarboxylate dispersingpolymers; or 15% of surfactant of which 6% was LAS, 3% was AES and 6%was nonionic surfactants and further contained 47% builder consisting offatty acid, 22% zeolite A, carbonate and silicate, and 5%polycarboxylate dispersing polymers; or 21% of surfactant of which 8.1%was LAS, 6.5% was AS, 4.0% was non ionic surfactants, and 2.5% wascationic surfactants (DSDMAC) and further contained 64% builderconsisting of fatty acid, carbonate, zeolite A, silicates, and citrate,and also contained 2.7% of dispersing polymers; or 16.9% surfactantsincluding soap of which 11% was LAS and 5.9% non-ionic and 4.1% soap,and 63% builders.

In one aspect the detergent is a European Liquid Laundry Detergentcomprising the following ingredients: 27% of surfactant of which 16.9%was AS, 6.7% was nonionic surfactants, and 3.5% was cationic surfactants(DSDMAC) and further contained 18.7% builder consisting of fatty acid,carbonate, citrate, and boric acid.

In one aspect the detergent is a North American Laundry Liquid detergentcomprising the following ingredients: 23% of surfactant of which 16% wasAES, 5% was LAS and 2% was non ionic surfactants. It further contained6% builder comprising soap, citric acid, DTPA and calcium formate; or23% of surfactant of which 16% was AES, 5% was LAS and 2% was non ionicsurfactants. It further contained 6% builder consisting of soap, citricacid, DTPA and calcium formate, and 5% poly-carboxylate dispersingpolymers.

In one aspect the detergent is a North American Laundry Powder detergentcomprising the following ingredients: 16.3% of surfactant of which 7.8%was LAS, 6.7% was AS and 1.8% was nonionic surfactants, and 60% buildercomprising fatty acid, zeolite A, carbonate and silicate; or 4.9% ofsurfactant of which 11.5% was LAS and 3.4% was non ionic surfactants,and 55% builder comprising fatty acid, zeolite A, carbonate andsilicate; or 19.5% of surfactant of which 4.5% was LAS, 13% was AS and2% was non ionic surfactants, and 61% builder comprising fatty acid,zeolite A, carbonate and silicate.

In one aspect the detergent is a Japanese laundry powder detergentcomprising the following ingredients: 24.3% of surfactant of which 11.1%was LAS, 11.6% was ester sulfonate and 1.6% was nonionic surfactants,and 60% builder comprising fatty acid, zeolite A, carbonate andsilicate; or 27.9% of surfactant of which 15 27.5% was LAS and 0.4% wasnonionic surfactants, and 64% builder comprising zeolite A, carbonate,citrate, phosphates and silicate.

In one aspect the detergent is a European color compact laundry powdercomprising the following ingredients: 21.1% of a surfactant system, ofwhich 8.1% was LAS, 6.5% was AS, 2.5% was Arguat 2T-70, and 4% wasnon-ionic surfactants, and 64% builder comprising fatty acid, zeolite A,carbonate, citric acid and silicate. The surfactant system was preparedseparately from the builder. The surfactant system was prepared eitherNeodol25-7 or Lutensol ON60 as nonionic surfactant.

The cleaning compositions of the present disclosure are advantageouslyemployed for example, in laundry applications, hard surface cleaning anddishwashing applications.

In some aspects, due to the unique advantage of increased performance atlow temperature, the lipolytic enzyme variants described herein areideally suited for laundry applications. Furthermore, the lipolyticenzyme variants of the present disclosure find use in granular andliquid compositions.

The lipolytic enzyme variants of the present disclosure also find use incleaning additive products. Such additive products are intended tosupplement and/or boost the performance of conventional detergentcompositions and can be added at any stage of the cleaning process.

In some embodiments, the present disclosure provides cleaning additiveproducts including at least one enzyme of the present disclosure that issuited for inclusion in a wash process when additional bleachingeffectiveness is desired. In some embodiments, the additive product isin its simplest form, one or more lipolytic enzymes. In someembodiments, the additive is packaged in dosage form for addition to acleaning process. In some embodiments, the additive is packaged indosage form for addition to a cleaning process where a source ofperoxygen is employed and increased bleaching effectiveness is desired.Any suitable single dosage unit form finds use with the presentdisclosure, including but not limited to pills, tablets, gelcaps, orother single dosage units such as pre-measured powders or liquids. Insome embodiments, filler(s) or carrier material(s) are included toincrease the volume of such compositions. Suitable filler or carriermaterials include, but are not limited to, various salts of sulfate,carbonate and silicate as well as talc, clay and the like. Suitablefiller or carrier materials for liquid compositions include, but are notlimited to water or low molecular weight primary and secondary alcoholsincluding polyols and diols. Examples of such alcohols include, but arenot limited to, methanol, ethanol, propanol and isopropanol. In someembodiments, the compositions contain from about 5% to about 90% of suchmaterials. Acidic fillers find use to reduce pH. Alternatively, in someembodiments, the cleaning additive includes adjunct ingredients, as morefully described below.

The present cleaning compositions and cleaning additives can comprise atleast one of the lipolytic enzyme variants provided herein, alone or incombination with other lipolytic enzymes and/or additional enzymes.

In some aspects, the cleaning compositions herein are formulated suchthat, during use in aqueous cleaning operations, the wash water willhave a pH of from about 5.0 to about 11.5, or about 6.0 to 8.0 or evenfrom about 7.5 to about 10.5. Liquid product formulations are typicallyformulated to have a neat pH from about 3.0 to about 9.0 or even fromabout 3 to about 8. Granular laundry products are typically formulatedto have a pH from about 6 to about 11, or even from about 8 to about 10.Techniques for controlling pH at recommended usage levels include theuse of buffers, alkalis, acids, etc., and are well known to thoseskilled in the art.

Suitable “low pH cleaning compositions” typically have a neat pH of fromabout 3 to about 8, and are typically free of surfactants that hydrolyzein such a pH environment. Such surfactants include sodium alkyl sulfatesurfactants that comprise at least one ethylene oxide moiety or evenfrom about 1 to about 16 moles of ethylene oxide. Such cleaningcompositions typically comprise a sufficient amount of a pH modifier,such as sodium hydroxide, monoethanolamine or hydrochloric acid, toprovide such cleaning composition with a neat pH of from about 3 toabout 8. Such compositions typically comprise at least one acid stableenzyme. In some embodiments, the compositions are liquids, while inother embodiments, they are solids. The pH of such liquid compositionsis typically measured as a neat pH. The pH of such solid compositions ismeasured as a 10% solids solution of said composition wherein thesolvent is distilled water. In these embodiments, all pH measurementsare taken at 20° C., unless otherwise indicated.

In some embodiments, when the lipolytic enzyme variant(s) is/areemployed in a granular composition or liquid, it is desirable for thelipolytic enzyme variant to be in the form of an encapsulated particleto protect the lipolytic enzyme variant from other components of thegranular composition during storage. In addition, encapsulation is alsoa means of controlling the availability of the lipolytic enzyme variantduring the cleaning process. In some embodiments, encapsulation enhancesthe performance of the lipolytic enzyme variant(s) and/or additionalenzymes. In this regard, the lipolytic enzyme variants of the presentdisclosure are encapsulated with any suitable encapsulating materialknown in the art. In some embodiments, the encapsulating materialtypically encapsulates at least part of the catalyst for the lipolyticenzyme variant(s) of the present disclosure. Typically, theencapsulating material is water-soluble and/or water-dispersible. Insome embodiments, the encapsulating material has a glass transitiontemperature (Tg) of 0° C. or higher. Glass transition temperature isdescribed in more detail in WO 97/11151. The encapsulating material istypically selected from consisting of carbohydrates, natural orsynthetic gums, chitin, chitosan, cellulose and cellulose derivatives,silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol,paraffin waxes, and combinations thereof. When the encapsulatingmaterial is a carbohydrate, it is typically selected frommonosaccharides, oligosaccharides, polysaccharides, and combinationsthereof. In some typical embodiments, the encapsulating material is astarch (See e.g., EP 0 922 499; U.S. Pat. Nos. 4,977,252; 5,354,559, and5,935,826). In some embodiments, the encapsulating material is amicrosphere made from plastic such as thermoplastics, acrylonitrile,methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixturesthereof; commercially available microspheres that find use include, butare not limited to those supplied by EXPANCEL® (Stockviksverken,Sweden), and PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®,LUXSIL®, Q-CEL®, and SPHERICEL® (PQ Corp., Valley Forge, Pa.).

As described herein, the lipolytic enzyme variants of the presentdisclosure find particular use in the cleaning industry, including, butnot limited to laundry and dish detergents. These applications placeenzymes under various environmental stresses. The lipolytic enzymevariants of the present disclosure provide advantages over manycurrently used enzymes, due to their stability under various conditions.

Indeed, there are a variety of wash conditions including varyingdetergent formulations, wash water volumes, wash water temperatures, andlengths of wash time, to which lipolytic enzymes involved in washing areexposed. In addition, detergent formulations used in differentgeographical areas have different concentrations of their relevantcomponents present in the wash water. For example, European detergentstypically have about 2000-10000 ppm of detergent components in the washwater, while Asian detergents typically have approximately 300-2500 ppmof detergent components in the wash water. In North America,particularly the United States, detergents typically have about 300ppm-1500 ppm of detergent components present in the wash water.

A high detergent concentration system includes detergents where greaterthan about 2000 ppm of the detergent components are present in the washwater. European detergents are generally considered to be high detergentconcentration systems as they have approximately 2000-10000 ppm ofdetergent components in the wash water.

Latin American detergents are generally high suds phosphate builderdetergents and the range of detergents used in Latin America can fall inboth the medium and high detergent concentrations as they range from1500 ppm to 6000 ppm of detergent components in the wash water. Asmentioned above, Brazil typically has approximately 1500 ppm ofdetergent components present in the wash water. However, other high sudsphosphate builder detergent geographies, not limited to other LatinAmerican countries, may have high detergent concentration systems up toabout 6000 ppm of detergent components present in the wash water.

Model detergent compostions useful for testing the lipolytic enzymevariant performance, such as but not limiting to, testing thethermostability of the lipolytic enzyme variant compared to a parentlipolytic enzyme include a model HDL composition comprising thefollowing ingredients: 3.1% lauryl alcohol ethoxylate (6EO), 9% sodiumlauryl ether sulphate, 3% sodium citrate, 0.8% sorbitol, 0.8% glycerol,0.5% triethanolamine, 1% ethanol (absolute); 7.6% linearalkylbenzenesulfonate (LAS), 3.0% potassium cocoate, 2.5% propyleneglycol, 0.01% 2-methyl-4-isothiazolin-3-one, 5.8% 4M NaOH and 62.9%demineralized water.

For North American (NA) and Western European (WE) heavy duty liquidlaundry (HDL) detergents, heat inactivation of the enzymes present incommercially-available detergents can be performed by placingpre-weighed liquid detergent (in a glass bottle) in a water bath at 95°C. for 2 hours or at 80° C. for 8 hours. The incubation time for heatinactivation of NA and WE auto dish washing (ADW) detergents is 8 hours.Both un-heated and heated detergents are assayed within 5 minutes ofdissolving the detergent to accurately determine percentage deactivated.For testing of enzyme activity in heat-inactivated detergents, workingsolutions of detergents are made from the heat inactivated stocks.

Appropriate amounts of water hardness (e.g., 6 gpg or 12 gpg or 17 gpgor 21 gpg) and buffer are added to the detergent solutions to match thedesired conditions. The solutions are mixed by vortexing or invertingthe bottles. Tables 12-13 provides information regarding some of thecommercially-available detergents and test conditions that can be used.In some experiments, additional and/or other commercially availabledetergents find use in the following Examples.

TABLE 12 Example of Laundry and Dish Washing Conditions for NorthAmerica, Japan and Western Europe. Laundry and Dish Washing ConditionsRegion Form Dose Detergent Buffer Gpg pH T (° C.) Laundry (Heavy DutyLiquid and Granular) NA HDL 0.78 g/l P&G TIDE ® 2X 5 mM HEPES  6  8.0 20WE HDL  5.0 g/L Henkel PERSIL ™ 5 mM HEPES 12  8.2 40 WE HDG  8.0 g/LP&G ARIEL ® 2 mM Na₂ CO₃ 12 10.5 40 JPN HDG  0.7 g/L P&G TIDE ® 2 mM Na₂CO₃  6 10.0 20 NA HDG  1.0 g/L P&G TIDE ® 2 mM Na₂ CO₃  6 10.0 20Automatic Dish Washing WE ADW  3.0 g/L RB CALGONIT ™ 2 mM Na₂ CO₃ 2110.0 40 NA ADW  3.0 g/L P&G CASCADE ® 2 mM Na₂ CO₃  9 10.0 40

TABLE 13 Example of Working Detergent Solutions Working DetergentSolutions Temp Detergent Detergent (C.) g/L pH Buffer Gpg TIDE ® 2X Cold16 0.98 8 5 mM 6 HEPES TIDE ® 2X Cold 32 0.98 8 5 mM 6 HEPES TIDE ® 2XCold 16 0.98 7 5 mM 6 MOPS

Table 14 provides granular laundry detergent compositions produced inaccordance with the disclosure suitable for laundering fabrics.

TABLE 14 Granular Laundry Detergent Compositions and Their ComponentsGranular Laundry Detergent Compositions and Their Components DetergentCompositions Component 1 2 3 4 5 6 Linear alkylbenzenesulfonate 15 12 2010 12 13 with aliphatic carbon chain length C₁₁-C₁₂ Other surfactants1.6 1.2 1.9 3.2 0.5 1.2 Phosphate builder(s) 2 3 4 Zeolite 1 1 4 1Silicate 4 5 2 3 3 5 Sodium Carbonate 2 5 5 4 0 3 Polyacrylate (MW 4500)1 0.6 1 1 1.5 1 Carboxymethyl cellulose 1 — 0.3 — 1.1 — (Finnfix BDA exCPKelco) Celluclean ® (15.6 mg/g) 0.23 0.17 0.5 0.2 0.2 0.6 Lipase (20mg/g) 0.2 0.1 0.3 Stainzyme Plus ® (14 mg/g) 0.23 0.17 0.5 0.2 0.2 0.6Mannaway 4.0T (4 mg/g) 0.1 0.1 0.1 Fluorescent Brightener(s) 0.16 0.060.16 0.18 0.16 0.16 Diethylenetriamine 0.6 0.6 0.25 0.6 0.6 pentaaceticacid or Ethylene diamine tetraacetic acid MgSO₄ 1 1 1 0.5 1 1 Bleach orBleach activator(s) 6.88 6.12 2.09 1.17 4.66 Ethoxylated thiopheneHueing 0.002 0.001 0.003 0.003 — — Dye⁵ Direct Violet 9 ex Ciba 0.00060.0004 0.0006 Specialty Chemicals Sulfate/Citric Acid/Sodium Balance to100% Bicarbonate/Moisture/perfume

In Table 14, all enzyme levels expressed as 00 enzyme raw material,except for lipolytic enzyme (of this disclosure) which is expressed as00 of active protein added to the product.

Table 15 provides granular laundry detergent compositions suitable fortop-loading automatic washing machines (detergent compositions 7-9) andfront loading washing machines 7 (detergent compositions 10-11). Thelipolytic enzyme variant tested and/or lipolytic enzyme of the presentdisclosure can be added separately to these formulations so that thefinal concentration in the wash liquor is between 0.01 ppm and 10 ppm.

TABLE 15 Granular Laundry Detergent Compositions and Their ComponentsGranular Laundry Detergent Compositions and Their Components DetergentComposition Component 7 8 9 10 11 Surfactants C₁₆₋₁₇ Branched alkyl 3.5515.8 sulfate C₁₂₋₁₄ alkyl sulphate 1.5 Sodium linear 9.6 10.6 7.5 9alkylbenzene- sulfonate with aliphatic chain length C₁₁-C₁₂ SodiumC_(14/15) alcohol 1.15 2.88 ethoxy-3-sulfate Sodium C_(14/15) alkyl 2.37sulphate C_(14/15) alcohol ethoxylate 1.17 1 with average 7 moles ofethoxylation mono-C₈₋₁₀ alkyl mono- 0.45 hydroxyethyl di-methylquaternary ammonium chloride Di methyl hydroxyl- 0.18 ethyl laurylammonium chloride Zeolite A 13.9 4.7 0.01 2.9 1.8 Sodium Silicate1.6.ratio 4 0.2 4 4 Sodium Silicate 2.35.ratio 8 Citric Acid 2.5 1.4Sodium tripolyphosphate 5 Sodium Carbonate 24.1 30 16.9 24.4 21Nonanoyloxybenzene- 5.78 2.81 0.96 suplhonate Oxaziridinium-based 0.030.017 bleach booster Tetrasodium S,S,-ethyl 0.2 enediaminedisuccinateDiethylenetriamine penta 0.61 0.33 (methylene phosphonic acid),heptasodium salt Hydroxyethane 0.29 0.45 dimethylene phosphonic acidEthylene diamine 0.27 tetraacetate MgSO4 0.47 0.5994 0.782 SodiumPercarbonate 7 4.4 15.9 19.1 Tetra Acetyl Ethylene 3.3 4.6 DiamineSodium Perborate 1.2 Monohydrate Carboxymethyl 0.1 0.17 1.69 0.23cellulose (e.g. Finnfix BDA ex CPKelco) Sodium Acrylic 0.0236 3.8 2 2.5acid/maleic acid co-polymer (70/30) Sodium polyacrylate 4 0.84 (SokalanPA30 CL) Terephthalate polymer 0.23 Polyethylene glycol/ 0.89 0.89 0.91vinyl acetate random graft co polymer Photobleach-zinc 0.005 0.001 0.002phthalocyanine tetrasulfonate C.I.Fluorescent 0.11 0.15 0.04 0.23 0.15Brightener 260 C.I.Fluorescent 0.1 Brightener 351 (Tinopal ® CBS) Sudssuppressor granule 0.25 0.07 0.04 Hydrophobically 0.019 0.028 modifiedcarboxy methyl cellulose (Finnifix ® SH-1) Bentonite 8.35 Miscellaneous(Dyes, Balance Balance Balance Balance Balance perfumes, process aids,moisture and sodium sulphate)

In Table 15, surfactant ingredients can be obtained from any suitablesupplier, including but not limited to BASF (e.g., LUTENSOL®), ShellChemicals, Stepan, Huntsman, and Clariant (e.g., PRAEPAGEN®). Zeolitecan be obtained from sources such as Industrial Zeolite. Citric acid andsodium citrate can be obtained from sources such as Jungbunzlauer.Sodium percarbonate, sodium carbonate, sodium bicarbonate and sodiumsesquicarbonate can be obtained from sources such as Solvay.Acrylate/maleate copolymers can be obtained from sources such as BASF.Carboxymethylcellulose and hydrophobically modified carboxymethylcellulose can be obtained from sources such as CPKelco. C.I. FluorescentBrightener 260 can be obtained from 3V Sigma (e.g., OPTIBLANC®,OPTIBLANC® 2M/G, OPTIBLANC® 2MG/LT Extra, or OPTIBLANC® Ecobright.Tetrasodium S,5S-ethyl enedi amine disuccinate can be obtained fromsources such as Innospec. Terephthalate co-polymer can be obtained fromClariant (e.g., REPELOTEX SF 2). In addition,1-Hydroxyethane-1,1-diphosphonic acid can be obtained from Thermphos.Oxaziridinium-based bleach booster has the following structure, whereR1=2-butyloctyl, and was produced according to US 2006/0089284A1.

The enzymes NATALASE®, TERMAMYL®, STAINZYME PLUS®, CELLUCLEAN® andMANNAWAY®, can be obtained from Novozymes. Zinc phthalocyaninetetrasulfonate can be obtained from Ciba Specialty Chemicals (e.g.,TINOLUX® BMC). Suds suppressor granule can be obtained from Dow Corning.In these detergent compositions, random graft copolymer is a polyvinylacetate grafted polyethylene oxide copolymer having a polyethylene oxidebackbone and multiple polyvinyl acetate side chains. The molecularweight of the polyethylene oxide backbone is about 6000 and the weightratio of the polyethylene oxide to polyvinyl acetate is about 40 to 60and no more than 1 grafting point per 50 ethylene oxide units.

Tables 16-18 provide additional granular detergent compositions suitablefor washing machines (detergents 36a-n). The lipolytic enzyme of thepresent disclosure is added separately to these formulations.

TABLE 16 Additional Granular Laundry Detergent Compositions and TheirComponents Additional Granular Laundry Detergent Compositions and TheirComponents Detergent Composition Component 36a 36b 36c 36d 36eSurfactants C₁₀ Nonionic 0.1843 C₁₆₋₁₇ Branched alkyl sulfate 3.53 3.533.53 C₁₂₋₁₄ alkyl sulphate Sodium linear alkylbenzene- 8.98 8.98 8.9813.58 14.75 sulfonate with aliphatic chain length C₁₁-C₁₂ SodiumC_(14/15) alcohol 1.28 1.28 1.28 ethoxy-3-sulfate Sodium C_(14/15) alkylsulphate 2.36 2.36 2.36 C_(14/15) alcohol ethoxylate with average 7moles of ethoxylation mono-C₈₋₁₀ alkyl mono- hydroxyethyl di-methylquaternary ammonium chloride Di methyl hydroxyl ethyl lauryl 0.1803ammonium chloride Zeolite A 15.31 15.31 15.31 4.47 Bentonite 8.35 SodiumSilicate 1.6.ratio 0.16 Sodium Silicate 2.0.ratio 3.72 3.72 3.72 8.41Sodium Silicate 2.35.ratio Citric Acid 0.0066 Sodium tripolyphosphate5.06 Sodium Carbonate 26.1 26.18 26.1 15.9 29.0Nonanoyloxybenzenesuplhonate 5.78 5.78 5.78 1.17 1.86Oxaziridinium-based bleach booster 0.037 0.037 0.037 Tetrasodium S,S,-ethylenediaminedisuccinate Diethylenetriamine penta (methylene 0.62 0.620.62 phosphonic acid), heptasodium salt Hydroxyethane dimethylenephosphonic acid Ethylene diamine tetraacetate 0.2701 MgSO4 0.056 0.0560.056 0.47 Sodium Percarbonate 7.06 7.06 3.64 Tetra Acetyl EthyleneDiamine Sodium Perborate Monohydrate 1.47 Carboxymethyl cellulose 0.380.38 0.38 0.173 (e.g. Finnfix BDA ex CPKelco) Sodium Acrylic acid/maleic3.79 3.78 3.79 3.64 acid co-polymer (70/30) Sodium polyacrylate 3.783.78 3.78 0.842 (Sokalan PA30 CL) Terephthalate polymer Polyethyleneglycol/vinyl acetate 0.89 random graft co polymer Photobleach-zincphthalocyanine tetrasulfonate C.I.Fluorescent Brightener 260 0.11250.1125 0.1125 0.043 0.15 C.I.Fluorescent Brightener 351 0.0952(Tinopal ® CBS) Suds suppressor granule 0.015 0.015 0.015 0.031Hyrdophobically modified carboxy methyl cellulose (Finnifix ® SH-1)Bentonite Miscellaneous (Dyes, perfumes, Balance Balance Balance BalanceBalance process aids, moisture and sodium sulphate)

TABLE 17 Additional Granular Laundry Detergent Compositions and TheirComponents Detergent Composition Component 36f 36g 36h 36i 36jSurfactants C₁₀ Nonionic 0.1142 0.2894 0.1885 0.1846 0.1885 C₁₆₋₁₇Branched alkyl sulfate C₁₂₋₁₄ alkyl sulphate Sodium linear 12.94 15.699.01 8.42 9.51 alkylbenzenesulfonate with aliphatic chain length C₁₁-C₁₂Sodium C_(14/15) alcohol ethoxy-3- sulfate Sodium C_(14/15) alkylsulphate C_(12/14) alcohol ethoxylate with 2.9 average 7 moles ofethoxylation C_(12/14) alcohol ethoxylate with 2.44 average 3 moles ofethoxylation C_(14/15) alcohol ethoxylate with 0.97 1.17 0.97 average 7moles of ethoxylation mono-C₈₋₁₀ alkyl mono-hydroxy 0.45 ethyl di-methylquaternary ammonium chloride Di methyl hydroxyl ethyl lauryl 0.195 0.45ammonium chloride Zeolite A 2.01 0.39 1.83 2.58 0.59 Sodium Silicate1.6.ratio 4.53 5.62 4.53 Sodium Silicate 2.0.ratio 10.1 Sodium Silicate2.35.ratio 7.05 Citric Acid 1.4 1.84 1.0 Sodium tripolyphosphate 5.73Sodium Carbonate 12.65 15.93 21.0 27.31 20.2Nonanoyloxybenzenesuplhonate 1.73 Oxaziridinium-based bleach 0.01680.0333 0.024 booster Tetrasodium S,S,- ethylenediaminedisuccinateDiethylenetriamine penta 0.327 0.3272 (methylene phosphonic acid),heptasodium salt Hydroxyethane dimethylene 0.45 0.2911 0.45 phosphonicacid Ethylene diamine tetraacetate 0.28 0.1957 MgSO4 0.54 0.79 0.64940.793 Sodium Percarbonate 19.1 15.85 22.5 Tetra Acetyl Ethylene Diamine4.554 3.71 5.24 Sodium Perborate Monohydrate 5.55 Carboxymethylcellulose 0.62 0.21 0.23 1.07 0.2622 (e.g. Finnfix BDA ex CPKelco)Sodium Acrylic acid/maleic 0.40 2.61 2.5 2.00 1.75 acid co-polymer(70/30) Sodium polyacrylate 0.0055 0.011 0.008 (Sokalan PA30 CL)Terephthalate polymer 0.231 Polyethylene glycol/vinyl acetate 0.55 1.400.911 0.8924 0.911 random graft co polymer Photobleach-zincphthalocyanine tetrasulfonate C.I.Fluorescent Brightener 260 0.11740.048 0.1455 0.2252 0.1455 C.I.Fluorescent Brightener 351 0.1049(Tinopal ® CBS) Suds suppressor granule 0.04 0.0658 0.04 Hyrdophobicallymodified carboxy methyl cellulose (Finnifix ® SH-1) BentoniteMiscellaneous (Dyes, perfumes, Balance Balance Balance Balance Balanceprocess aids, moisture and sodium sulphate)

TABLE 18 Additional Granular Laundry Detergent Compositions and TheirComponents Detergent Composition Component 36k 36l 36m 36n SurfactantsC₁₀ Nonionic 0.1979 0.1979 0.1979 0.1979 C₁₆₋₁₇ Branched alkyl sulfateC₁₂₋₁₄ alkyl sulphate Sodium linear alkylbenzenesulfonate 8.92 8.92 11.511.5 with aliphatic chain length C₁₁-C₁₂ Sodium C_(14/15) alcohol 1.621.62 1.125 1.125 ethoxy-3-sulfate Sodium C_(14/15) alkyl sulphateC_(14/15) alcohol ethoxylate with 1.0 1.0 1.5 1.5 average 7 moles ofethoxylation mono-C₈₋₁₀ alkyl mono-hydroxy ethyl di-methyl quaternaryammonium chloride Di methyl hydroxyl ethyl lauryl ammonium chlorideZeolite A 1.63 1.63 2.0 2.0 Sodium Silicate 1.6.ratio 4.75 4.75 4.754.75 Sodium Silicate 2.0.ratio 0.06 0.06 Sodium Silicate 2.35.ratioCitric Acid 1.10 1.10 1.1 1.1 Sodium tripolyphosphate Sodium Carbonate23.3 23.3 23.3 23.3 Nonanoyloxybenzenesuplhonate Oxaziridinium-basedbleach booster 0.021 0.021 0.015 0.015 Tetrasodium S,S,- 0.26 0.26 0.260.26 ethylenediaminedi succinate Diethylenetriamine penta (methylenephosphonic acid), heptasodium salt Hydroxy ethane dimethylene 0.47 0.470.47 0.47 phosphonic acid Ethylene diamine tetraacetate MgSO4 0.83 0.830.82 0.82 Sodium Percarbonate 19.35 19.35 19.35 19.35 Tetra AcetylEthylene Diamine 4.51 4.51 4.51 4.51 Sodium Perborate MonohydrateCarboxymethyl cellulose 1.01 1.01 1.01 1.01 (e.g. Finnfix BDA exCPKelco) Sodium Acrylic acid/maleic 1.84 1.84 1.84 1.84 acid co-polymer(70/30) Sodium polyacrylate 0.007 0.007 0.005 0.005 (Sokalan PA30 CL)Terephthalate polymer 0.179 0.179 0.179 0.179 Polyethylene glycol/vinylacetate 0.96 0.96 0.96 0.96 random graft co polymer Photobleach-zincphthalocyanine tetrasulfonate C.I.Fluorescent Brightener 260 0.153 0.1530.171 0.171 C.I.Fluorescent Brightener 351 (Tinopal ® CBS) Sudssuppressor granule 0.042 0.042 0.042 0.042 Hyrdophobically modifiedcarboxy methyl cellulose (Finnifix ® SH-1) Bentonite Miscellaneous(Dyes, perfumes, Balance Balance Balance Balance Balance process aids,moisture and sodium sulphate)Notes for detergent compositions 36 a-n in Tables 16, 17, and 18:Surfactant ingredients can be obtained from BASF, Ludwigshafen, Germany(Lutensol®); Shell Chemicals, London, UK; Stepan, Northfield, Ill., USA;Huntsman, Huntsman, Salt Lake City, Utah, USA; Clariant, Sulzbach,Germany (Praepagen®).Zeolite can be obtained from Industrial Zeolite (UK) Ltd, Grays, Essex,UK.Citric acid and sodium citrate can be obtained from Jungbunzlauer,Basel, Switzerland.Sodium percarbonate, sodium carbonate, sodium bicarbonate and sodiumsesquicarbonate can be obtained from Solvay, Brussels, Belgium.Acrylate/maleate copolymers can be obtained from BASF, Ludwigshafen,Germany.Carboxymethylcellulose and hydrophobically modified carboxymethylcellulose can be obtained from CPKelco, Arnhem, The Netherlands.C.I. Fluorescent Brightener 260 can be obtained from 3V Sigma, Bergamo,Italy as Optiblanc® Optiblanc® 2M/G, Optiblanc® 2MG/LT Extra, orOptiblanc® Ecobright.Tetrasodium S,S-ethylenediamine disuccinate can be obtained fromInnospec, Ellesmere Port, UK.Terephthalate co-polymer can be obtained from Clariant under thetradename Repelotex SF 2. 1-Hydroxyethane-1,1-diphosphonic acid can beobtained from Thermphos, Vlissingen-Oost, The Netherlands.Oxaziridinium-based bleach booster has the following structure, whereR1=2-butyloctyl, and was produced according to US 2006/0089284A1.

Enzymes Natalase®, Termamyl®, Stainzyme Plus®, Celluclean® andMannaway®, can be obtained from Novozymes, Bagsvaerd, Denmark.Zinc phthalocyanine tetrasulfonate can be obtained from Ciba SpecialtyChemicals, Basel, Switzerland, as Tinolux® BMC.Suds suppressor granule can be obtained from Dow Corning, Barry, UK.Random graft copolymer is a polyvinyl acetate grafted polyethylene oxidecopolymer having a polyethylene oxide backbone and multiple polyvinylacetate side chains. The molecular weight of the polyethylene oxidebackbone is about 6000 and the weight ratio of the polyethylene oxide topolyvinyl acetate is about 40 to 60 and no more than 1 grafting pointper 50 ethylene oxide units.

In light of the foregoing, it is evident that concentrations ofdetergent compositions in typical wash solutions throughout the worldvaries from less than about 300 ppm of detergent composition (“lowdetergent concentration geographies”) to 10000 ppm in Europe and about6000 ppm in high suds phosphate builder geographies.

The concentrations of the typical wash solutions are determinedempirically. For example, in the U.S., a typical washing machine holds avolume of about 64.4 L of wash solution. Accordingly, in order to obtaina concentration of about 1000 ppm of detergent within the wash solutionabout 64.4 g of detergent composition must be added to the 64.4 L ofwash solution. This amount is the typical amount measured into the washwater by the consumer using the measuring cup provided with thedetergent.

As a further example, different geographies use different washtemperatures. The temperature of the wash water in Japan is typicallyless than that used in Europe. For example, the temperature of the washwater in North America and Japan is typically between about 10 and about30° C. (e.g., about 20° C.), whereas the temperature of wash water inEurope is typically between about 30 and about 60° C. (e.g., about 40°C.). However, in the interest of saving energy, many consumers areswitching to using cold water washing. In addition, in some furtherregions, cold water is typically used for laundry, as well as dishwashing applications. In some embodiments, the “cold water washing” ofthe present disclosure utilizes “cold water detergent” suitable forwashing at temperatures from about 10° C. to about 40° C., or from about20° C. to about 30° C., or from about 15° C. to about 25° C., as well asall other combinations within the range of about 15° C. to about 35° C.,and all ranges within 10° C. to 40° C.

As a further example, different geographies typically have differentwater hardness. Water hardness is usually described in terms of thegrains per gallon mixed Ca²⁺/Mg²⁺ Hardness is a measure of the amount ofcalcium (Ca²⁺) and magnesium (Mg²⁺) in the water. Most water in theUnited States is hard, but the degree of hardness varies (Table 19).Moderately hard (60-120 ppm) to hard (121-181 ppm) water has 60 to 181parts per million (parts per million converted to grains per U.S. gallonis ppm # divided by 17.1 equals grains per gallon) of hardness minerals.

TABLE 19 Water hardness Water Grains per gallon Parts per million Softless than 1.0 less than 17 Slightly hard 1.0 to 3.5 17 to 60 Moderatelyhard 3.5 to 7.0  60 to 120 Hard  7.0 to 10.5 120 to 180 Very hardgreater than 10.5 greater than 180

European water hardness is typically greater than about 10.5 (forexample about 10.5 to 5 about 20.0) grains per gallon mixed Ca²⁺/Mg²⁺(e.g., about 15 grains per gallon mixed Ca²⁺/Mg²⁺). North American waterhardness is typically greater than Japanese water hardness, but lessthan European water hardness. For example, North American water hardnesscan be between about 3 to about 10 grains, about 3 to about 8 grains orabout 6 grains. Japanese water hardness is typically lower than NorthAmerican water hardness, usually less than about 4, for example about 3grains per gallon mixed Ca²⁺/Mg²⁺.

Accordingly, in some embodiments, the present disclosure provideslipolytic enzyme variants that show improved wash performance in atleast one set of wash conditions (e.g., water temperature, waterhardness, and/or detergent concentration). In some embodiments, thelipolytic enzyme variants of the present disclosure are comparable inwash performance to other lipase lipolytic enzymes. In some embodiments,the lipolytic enzyme variants of the present disclosure exhibit enhancedwash performance as compared to lipase lipolytic enzymes currentlycommercially available.

In addition, the lipolytic enzyme variants of the present disclosurefind use in cleaning compositions that do not include detergents, againeither alone or in combination with builders and stabilizers.

In some embodiments, an effective amount of one or more lipolytic enzymevariant(s) provided herein is included in compositions useful forcleaning a items in need of lipid stain removal. Such cleaningcompositions include cleaning compositions for such applications ascleaning laundry, hard surfaces (e.g., the hard surface of a table,table top, wall, furniture item, floor, ceiling, medical instrument,examination table, etc.), fabrics, and dishes. Indeed, in someembodiments, the present disclosure provides fabric cleaningcompositions, while in other embodiments, the present disclosureprovides non-fabric cleaning compositions. It is intended that thepresent disclosure encompass detergent compositions in any form (i.e.,liquid, granular, bar, semi-solid, gels, emulsions, tablets, capsules,etc.).

As indicated above, the cleaning compositions of the present disclosureare formulated into any suitable form and prepared by any process chosenby the formulator, non-limiting examples of which are described in U.S.Pat. Nos. 5,879,584, 5,691,297, 5,574,005, 5,569,645, 5,516,448,5,489,392, and 5,486,303, all of which are incorporated herein byreference. In some embodiments in which a low pH cleaning composition isdesired, the pH of such composition is adjusted via the addition of anacidic material such as HCl.

Methods of Use

In some embodiments, the cleaning compositions of the present disclosurefind use in cleaning surfaces (e.g., dishware), laundry, hard surfaces,contact lenses, fabric, etc. In some embodiments, at least a portion ofthe surface is contacted with at least one embodiment of the cleaningcompositions of the present disclosure, in neat form or diluted in awash liquor, and then the surface is optionally washed and/or rinsed.

For purposes of the present disclosure, “washing” includes, but is notlimited to, scrubbing, and mechanical washing. In some embodiments, thecleaning compositions of the present disclosure are used atconcentrations of from about 300 ppm to about 15,000 ppm in solution. Insome embodiments in which the wash solvent is water, the watertemperature typically ranges from about 5° C. to about 90° C.

The compositions of the present disclosure also find use in detergentadditive products in solid or liquid form. Such additive products areintended to supplement and/or boost the performance of conventionaldetergent compositions and can be added at any stage of the cleaningprocess.

The present disclosure provides methods for cleaning or washing an itemor surface (e.g., hard surface) in need of cleaning, including, but notlimited to methods for cleaning or washing a dishware item, a tablewareitem, a fabric item, a laundry item, personal care item, etc., or thelike, and methods for cleaning or washing a hard or soft surface (e.g.,a hard surface of an item).

In some embodiments, the present disclosure provides methods for washingincluding manual dishwashing, hard surface cleaning and automaticdishwashing.

In some embodiments, the present disclosure provides a method forcleaning an item, object, or surface in need of cleaning, the methodcomprising contacting the item or surface (or a portion of the item orsurface desired to be cleaned) with at least one variant lipaselipolytic enzyme of the present disclosure or a composition of thepresent disclosure for a sufficient time and/or under conditionssuitable and/or effective to clean the item, object, or surface to adesired degree. Some such methods further comprise rinsing the item,object, or surface with water. For some such methods, the cleaningcomposition is a dishwashing detergent composition and the item orobject to be cleaned is a dishware item or tableware item. As usedherein, a “dishware item” is an item generally used in serving or eatingfood. A dishware item can be, but is not limited to for example, a dish,plate, cup, bowl and the like. As used herein, “tableware” is a broaderterm that includes, but is not limited to for example, dishes, cutlery,knives, forks, spoons, chopsticks, glassware, pitchers, sauce boats,drinking vessels, serving items, etc. It is intended that “tablewareitem” includes any of these or similar items for serving or eating food.For some such methods, the cleaning composition is an automaticdishwashing detergent composition or a hand dishwashing detergentcomposition and the item or object to be cleaned is a dishware ortableware item. For some such methods, the cleaning composition is alaundry detergent composition (e.g., a power laundry detergentcomposition or a liquid laundry detergent composition), and the item tobe cleaned is a fabric item. In some other embodiments, the cleaningcomposition is a laundry pre-treatment composition.

In some embodiments, the present disclosure provides methods forcleaning or washing a fabric item optionally in need of cleaning orwashing, respectively. In some embodiments, the methods compriseproviding a composition comprising the lipolytic enzyme variant,including but not limited to fabric or laundry cleaning composition, anda fabric item or laundry item in need of cleaning, and contacting thefabric item or laundry item (or a portion of the item desired to becleaned) with the composition under conditions sufficient or effectiveto clean or wash the fabric or laundry item to a desired degree.

In some embodiments, the present disclosure provides a method forcleaning or washing an item or surface (e.g., hard surface including thehard surface of a table, table top, wall, furniture item, floor,ceiling, medical instrument, examination table, etc) optionally in needof cleaning, the method comprising providing an item or surface to becleaned or washed and contacting the item or surface (or a portion ofthe item or surface desired to be cleaned or washed) with at least onelipase variant of the disclosure or a composition of the disclosurecomprising at least one such lipase variant for a sufficient time and/orunder conditions sufficient or effective to clean or wash the item orsurface to a desired degree. Such compositions include, but are notlimited to for example, a cleaning composition or detergent compositionof the disclosure (e.g., a hand dishwashing detergent composition, handdishwashing cleaning composition, laundry detergent or fabric detergentor laundry or fabric cleaning composition, liquid laundry detergent,liquid laundry cleaning composition, powder laundry detergentcomposition, powder laundry cleaning composition, automatic dishwashingdetergent composition, laundry booster cleaning or detergentcomposition, laundry cleaning additive, and laundry pre-spottercomposition, etc.). In some embodiments, the method is repeated one ormore times, particularly if additional cleaning or washing is desired.For example, in some instance, the method optionally further comprisesallowing the item or surface to remain in contact with the at least onelipolytic enzyme variant or composition for a period of time sufficientor effective to clean or wash the item or surface to the desired degree.In some embodiments, the methods further comprise rinsing the item orsurface with water and/or another liquid. In some embodiments, themethods further comprise contacting the item or surface with at leastone lipolytic enzyme variant of the disclosure or a composition of thedisclosure again and allowing the item or surface to remain in contactwith the at least one lipolytic enzyme variant or composition for aperiod of time sufficient to clean or wash the item or surface to thedesired degree. In some embodiments, the cleaning composition is adishwashing detergent composition and the item to be cleaned is adishware or tableware item. In some embodiments of the present methods,the cleaning composition is an automatic dishwashing detergentcomposition or a hand dishwashing detergent composition and the item tobe cleaned is a dishware or tableware item. In some embodiments of themethods, the cleaning composition is a laundry detergent composition andthe item to be cleaned is a fabric item.

The present disclosure also provides methods of cleaning a tableware ordishware item in an automatic dishwashing machine, the method comprisingproviding an automatic dishwashing machine, placing an amount of anautomatic dishwashing composition comprising at least one lipase variantof the present disclosure or a composition of the disclosure sufficientto clean the tableware or dishware item in the machine (e.g., by placingthe composition in an appropriate or provided detergent compartment ordispenser in the machine), putting a dishware or tableware item in themachine, and operating the machine so as to clean the tableware ordishware item (e.g., as per the manufacturer's instructions). In someembodiments, the methods include any automatic dishwashing compositiondescribed herein, which comprises, but is not limited to at least onelipase variant provided herein. The amount of automatic dishwashingcomposition to be used can be readily determined according to themanufacturer's instructions or suggestions and any form of automaticdishwashing composition comprising at least one lipolytic enzyme variantof the disclosure (e.g., liquid, powder, solid, gel, tablet, etc.),including any described herein, may be employed.

The present disclosure also provides methods for cleaning a surface,item or object optionally in need of cleaning, the method comprisescontacting the item or surface (or a portion of the item or surfacedesired to be cleaned) with at least one variant lipase of the presentdisclosure or a cleaning composition of the disclosure in neat form ordiluted in a wash liquor for a sufficient time and/or under conditionssufficient or effective to clean or wash the item or surface to adesired degree. The surface, item, or object may then be (optionally)washed and/or rinsed if desired. For purposes of the present disclosure,“washing” includes, but is not limited to for example, scrubbing andmechanical agitation. In some embodiments, the cleaning compositions areemployed at concentrations of from about 500 ppm to about 15,000 ppm insolution (e.g., aqueous solution). When the wash solvent is water, thewater temperature typically ranges from about 5° C. to about 90° C. andwhen the surface, item or object comprises a fabric, the water to fabricmass ratio is typically from about 1:1 to about 30:1.

The present disclosure also provides methods of cleaning a laundry orfabric item in an washing machine, the method comprising providing anwashing machine, placing an amount of a laundry detergent compositioncomprising at least one variant lipase of the disclosure sufficient toclean the laundry or fabric item in the machine (e.g., by placing thecomposition in an appropriate or provided detergent compartment ordispenser in the machine), placing the laundry or fabric item in themachine, and operating the machine so as to clean the laundry or fabricitem (e.g., as per the manufacturer's instructions). The methods of thepresent disclosure include any laundry washing detergent compositiondescribed herein, comprising but not limited to at least one of anyvariant lipase provided herein. The amount of laundry detergentcomposition to be used can be readily determined according tomanufacturer's instructions or suggestions and any form of laundrydetergent composition comprising at least one lipolytic enzyme variantof the disclosure (e.g., solid, powder, liquid, tablet, gel, etc.),including any described herein, may be employed.

The present disclosure also provides variants, such as lipase enzymevariants useful for pulp and paper processing, including controllingorganic contaminants in fibers. The fiber can be cellulose fibers and insome instances are recycled fibers from a variety of paper products orfiber containing products, such as old corrugated containers (OCC), oldnewsprint (ONP), mixed office waste (MOW), or combinations thereof.These types of paper containing products typically contain large amountsof organic contaminants which are present in the paper products. Whenthese types of paper products are recycled, these organic contaminantsare present along with the fibers formed during the pulping stage of apapermaking process. These organic contaminants, if not substantiallyremoved, can severely interfere with subsequent stages in thepapermaking process by affecting the quality of the resulting sheets ofpaper formed and/or affecting the machinery used to form the paper.Accordingly, the removal of such organic contaminants is important tothe paper making process when such organic contaminants are present infibers.

For purposes of the present disclosure, examples of organic contaminantsinclude what is known in the industry as “stickies” and include, but arenot limited to, synthetic polymers resulting from adhesives and thelike, glues, hot melts, coatings, coating binders, ink residues,de-inking chemicals, wood resins, rosin, and unpulped wet strengthresins. These types of materials are typically found in paper containingproducts, such as newsprint, corrugated container, and/or mixed officewaste. These organic contaminants typically will have polymers present,such as styrene butadiene rubber, vinyl acrylates, polyisoprene,polybutadiene, natural rubber, ethyl vinyl acetates, polyvinyl acetates,ethylvinyl alcohols, polyvinyl alcohols, styrene acrylates, and othersynthetic type polymers.

In the process of the present disclosure, these organic contaminants arecontrolled by contacting the fiber containing the organic contaminantswith a composition containing at least one variant of the presentdisclosure for a sufficient time and in a sufficient amount to controlthe organic contaminants present in the fiber. The compositions of thepresent disclosure preferably disperse or convert the organiccontaminants to organic species that do not affect the paper makingprocess. For instance, the polyvinyl acetates are preferably dispersedand/or converted to polyvinyl alcohols, which do not affect thepapermaking process. This preferred manner that the compositions achievecontrol of organic contaminants is quite different from collectingcontaminants by flotation.

For purposes of the present disclosure, controlling organic contaminantspresent in fibers having organic contaminants is understood as one ormore of the following: reducing the size of contaminant particles,reducing the number or amount of measurable particles present, and/orreducing the tackiness of the organic contaminants. In some embodiments,when controlling organic contaminants using the methods of the presentdisclosure, all of these reductions occur. In some embodiments, thereduction of the size of contaminant particles is by at least about 5%,or by from about 10% to about 75% as compared to when no variant of thepresent disclosure is present. Similarly, the reduction in the number oramount of organic contaminants present in the fiber is reduced by atleast about 5%, or by from about 10% to about 75% when compared tofibers which have not been treated with a variant of the presentdisclosure. Also, the reduction of tackiness of the organic contaminantscan be reduced by at least about 5%, or by from about 10% to about 75%when compared to fibers which have not been treated with a variant ofthe present disclosure.

The compositions containing at least one variant of the presentdisclosure can also contain as an option other conventional papertreatment chemicals or ingredients such as, but not limited to,surfactants, solvents, suspension aids, fillers, chelants,preservatives, buffers, water, stabilizers, and the like. Theseadditional ingredients can be present in conventional amounts.

In some embodiments of the disclosure, a method is provided for treatingpolyester, including clean, unsoiled polyester, comprising contactingsaid polyester textile with an enzyme solution having variant of thepresent disclosure for a time and under conditions such that theproperties of the polyester are modified. Preferably, the polyester is afiber, yarn, fabric or finished textile product comprising such fiber,yarn or fabric. Further preferably, the properties that are modifiedcomprise those such as improved hand, feel and/or weight of a textilemade from such fiber, yarn or article. In some embodiments, the presentdisclosure is to provide for a mechanism to modify the textilecharacteristics of a polyester comprising textile. Thus, in thisembodiment of the disclosure, it is often advantageous to apply thepolyesterase to textile products which are unsoiled, i.e., do notcomprise stains which are typically subjected to commercial laundrydetergents. In other embodiments, the present disclosure is to providefor a method of laundering stains from polyester fabrics.

In another embodiment of the disclosure, a method is provided fortreating a polyester fiber, yarn or fabric, prior to its incorporationinto a textile product or the application of a textile finish with anenzyme variant of the present disclosure for a time and under conditionssuch that the properties of the polyester are modified. Accordingly, inthe embodiment wherein textile components are treated separately, thetreated polyester components (i.e., fibers, yarns, fabrics), can beincorporated into a textile product through standard methods forproducing polyester textiles, e.g., processes such as weaving, sewingand cutting and stitching, thus conferring the modifications to thefinished textile product.

In yet another method embodiment of the disclosure, a method is providedfor treating a polyester resin or film with an enzyme variant of thepresent disclosure for a time and under conditions such that theproperties of the polyester are modified. The treated polyester may be afinished resin or film product or may be incorporated into a productthrough, for example, mechanical construction, thus conferring themodifications to the finished textile product.

In yet another method embodiment of the disclosure, a polyester wasteproduct is treated with an enzyme variant of the present disclosure todegrade the polyester waste product to easily dispose of or recycledcompounds. This embodiment is particularly useful in the degradation ofpolyester based plastics which are becoming increasingly problematic inwaste disposal and dumping. An alternative of this embodiment is thatthe present disclosure may be used to increase the amount of microbiallydigestible material in a waste product so as to facilitate completedegradation or composting of such waste.

In the method according to the disclosure, the solution containing anenzyme variant of the present disclosure as provided herein is contactedwith the polyester fiber, yarn, fabric or textile which comprises suchfiber, yarn or fabric under conditions suitable for the enzyme toexhibit polyester modification. The present disclosure is preferablydirected to the use of the polyesterase in the manufacture of thetextile product, and not necessarily in combination with a detergent forthe purpose of removing stains which occur during wear. Thus, in thisembodiment, the application of the enzyme variant of the presentdisclosure to the polyester article occurs prior to spinning of thefiber into a yarn, prior to the incorporation of the yarn into a fabricand/or prior to the construction of the textile product which comprisesthe polyester. However, it is within the present disclosure as well, andalso a preferred embodiment hereon, to treat the completed textileproduct with the enzyme variant of the present disclosure identifiedherein.

Non-limiting examples of compositions and methods disclosed herein areas follows:1. A lipolytic enzyme variant or an active fragment thereof comprisingat least two amino acid modifications to a parent lipolytic enzyme,wherein a first amino acid modification is at a position of thelipolytic enzyme variant selected from the group consisting of 4, 11,19, 22, 26, 39, 53, 60, 64, 67, 70, 71, 93, 95, 111, 117, 119, 120, 121,125, 127, 131, 134, 141, 142, 143, 146, 149, 154, 159, 163, 166, 170,179, 185, 196, 199, 203, 204, 205, 206, 208, 209, 212, 217, 218, 221,224, 227, 229, 235, 247, 252, 261, 262, 265, 272, 280 and 286, whereinthe amino acid positions of the variant are numbered by correspondencewith the amino acid sequence of SEQ ID NO: 1, wherein the lipolyticenzyme variant has at least 60% sequence identity to the amino acidsequence SEQ ID NO: 1.2. The lipolytic enzyme variant or active fragment thereof of embodiment1, wherein the at least two amino acid modifications to a parentlipolytic enzyme are at a position of the lipolytic enzyme variantselected from the group consisting of 4, 11, 19, 22, 26, 39, 53, 60, 64,67, 70, 71, 93, 95, 111, 117, 119, 120, 121, 125, 127, 131, 134, 141,142, 143, 146, 149, 154, 159, 163, 166, 170, 179, 185, 196, 199, 203,204, 205, 206, 208, 209, 212, 217, 218, 221, 224, 227, 229, 235, 247,252, 261, 262, 265, 272, 280, and 286, wherein the amino acid positionsof the variant are numbered by correspondence with the amino acidsequence of SEQ ID NO: 1.2b. A lipolytic enzyme variant or an active fragment thereof consistingof two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen or fourteen amino acid modifications to a parent lipolyticenzyme, wherein the two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen or fourteen acid modifications is at aposition of the lipolytic enzyme variant selected from the groupconsisting of 4, 11, 19, 22, 26, 39, 53, 60, 64, 67, 70, 71, 93, 95,111, 117, 119, 120, 121, 125, 127, 131, 134, 141, 142, 143, 146, 149,154, 159, 163, 166, 170, 179, 185, 196, 199, 203, 204, 205, 206, 208,209, 212, 217, 218, 221, 224, 227, 229, 235, 247, 252, 261, 262, 265,272, 280 and 286, wherein the amino acid positions of the variant arenumbered by correspondence with the amino acid sequence of SEQ ID NO: 1.3. The lipolytic enzyme variant or an active fragment thereof ofembodiment 1 or 2, wherein said variant is derived from a parentlipolytic enzyme having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% aminoacid sequence identity to the amino acid sequence of SEQ ID NO: 1.4. The lipolytic enzyme variant or an active fragment thereof ofembodiment 1 or embodiment 2, wherein said variant comprises an aminoacid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequenceidentity to the amino acid sequence of SEQ ID NO: 1.5. The lipolytic enzyme variant or active fragment of claim 1 or 2,wherein the amino acid sequence has at least two or more of thefollowing features with respect to SEQ ID NO: 1: a K at position 4; an Rat position 11; an L at position 19; an F at position 22; a C atposition 26; an I at position 39; a Q at position 53; an L at position60; a V or T at position 64; a G at position 67; a V at position 70; anE at position 71; an S at position 93; a Q at position 95; a T atposition 111; a D, E, G, Q or S at position 117; a V at position 119; anE or Q or S or V at position 120; an E or S at position 121; an E atposition 125; a D or E at position 127; a D, E, K or T at position 131;a E or M at position 134; an N at position 141; a V at position 142; anL at position 143; a H at position 146; an I at position 149; a V atposition 154; a D at position 159; a G at position 163; a T at position166; an E or S at position 170; a K at position 179; a Q at position185; a L at position 196; an H at position 199; a C, K, N, R, S ort T atposition 203; a V or Y at position 204; a W at position 205; a C, E or Qat position 206; an E or Q at position 208; an A or D at position 209; aR at position 212; a Y at position 217; a D at position 218; a T atposition 221; an S at position 224; a D or V at position 227; an A, C,H, P, Q, S, T or V at position 229; an A at position 235; an R atposition 247; a D, G, Q or V at position 252; an A or L at positon 261;a C or M at position 262; an A at position 265; a K, P or V at position272; a K at position 280; and a Q at position 286, wherein the aminoacid positions of the variant are numbered by correspondence with theamino acid sequence of SEQ ID NO: 1.6. The lipolytic enzyme variant or an active fragment thereof of anypreceding embodiment, wherein the variant or active fragment thereofcomprises amino acid modifications selected from the group consisting ofY022F-L203R, A131D-L203R, Y127D-L203R, A131E-L203R, L203R-I204Y,L203R-G206C, L203R-K208Q, L203R-G209D, L203R-G209A, L203R-R229Q,L203R-E252Q, L203R-V261L, L203R-A262M, L203R-A262C, L203R-I272P,Y022F-R117D-L203R, Y022F-R117Q-L203R, Y022F-A131E-L203R,Y022F-L203R-G206C, Y022F-L203R-K208Q, Y022F-L203R-G209D,Y022F-L203R-G209A, Y022F-L203R-R229Q, Y022F-L203R-R229T,Y022F-L203R-I272P, R117D-A131E-L203R, R117D-L203R-E252Q,R117Q-A131D-L203R, A131D-L203R-G206C, A131D-L203R-R229T,A131D-L203R-E252Q, R117Q-L203R-K208Q, Y127D-L203R-K208Q,A131E-L203R-K208Q, L203R-I204Y-K208Q, L203R-K208Q-G206C,L203R-K208Q-G209D, L203R-K208Q-G209A, L203R-K208Q-R229Q,L203R-K208Q-R229T, L203R-K208Q-E252Q, L203R-K208Q-V261L,L203R-K208Q-A262M, L203R-K208Q-I272P, Y127D-L203R-G209D,A131E-L203R-G209D, L203R-I204Y-G209D, L203R-G206C-G209D,L203R-G209D-R229Q, L203R-G209D-R229T, L203R-G209D-E252Q,L203R-G209D-I272P, R117Q-L203R-R229T, Y127D-L203R-R229T,A131E-L203R-R229T, L203R-I204Y-R229T, L203R-G206C-R229T,L203R-G209A-R229T, L203R-R229T-E252Q, L203R-R229T-A262M,L203R-R229T-I272P, R117Q-L203R-I272P, Y127D-L203R-I272P,A131E-L203R-I272P, L203R-I204Y-I272P, L203R-G206C-I272P,L203R-G209A-I272P, L203R-E252Q-I272P, A131D-L203R-G209D-R229T,A131D-L203R-R229T-I272P, A131D-L203S-R229T-I272P,L203R-K208Q-G209D-R229T, L203R-K208Q-R229T-I272P,L203S-K208Q-G209D-R229T, L203S-K208Q-R229T-I272P,Y022F-R117Q-A131T-F143L-L203R-G209D-R229Q,Y199H-L203R-G209D-A217Y-R229Q, L203R-G209D-A217Y-R229Q,R117Q-L203R-G209D-R229C, Y199H-A217Y,A217Y-V221T-R229C-E252Q-A262M-I272P, E252Q-A262M-I272P,Y022F-A159D-L203R-G206C-K208Q-G209D-I272P,Y022F-L203R-G206C-K208Q-G209D, P125E-L203R-K208Q-G209D,Y022F-R229C-E252Q-I272P, A159D-L203R-G209D,T004K-K071E-R117D-A131T-F143L-N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229C-N280K,T004K-K071E-R117Q-A131D-F143L-N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229C-N280K,A159D-L203R-G209D-R229H, A159D-L203R-G209D-R229C,A159D-L203R-G209D-R229V, A159D-L203R-G206E-G209D,Y022F-R117Q-A131D-L203R-R229V-E252Q,Y022F-R053Q-R117Q-E252Q-V261L-I272P, Y022F-R117Q-A131D-E252Q,Y022F-R117Q-R229V-E252Q-V261L-I272P, Y022F-R053Q-R229V-E252Q-I272P,R117Q-A131D-E252Q-I272P, Y022F-R053Q-R117Q-R229V-V261L,Y022F-R117Q-G209D-R229V-E252Q-I272P, Y022F-A131D-R229V-E252Q-I272P,Y022F-R053Q-R117Q-L203R-R229V-E252Q-V261L, Y022F-R117Q-R229V,Y022F-R117Q-A131D-R229V-E252Q,Y022F-R053Q-R117Q-L203R-R229V-E252Q-V261L-I272P,Y022F-R053Q-R117Q-L203R-R229V-E252Q-I272P,Y022F-R053Q-R117Q-L203R-R229V-E252Q,Y022F-A131D-L203R-R229V-E252Q-I272P, Y022F-R053Q-R117Q-E252Q-I272P,Y022F-R117Q-E252Q-V261L, R117Q-A131D-L203R-R229V-E252Q,Y022F-R229V-E252Q-I272P, Y022F-R117Q-R229V-I272P, Y022F-R117D,Y022F-K134E-R229Q, Y022F-R117D-I272P, R117E-A131E-I204Y-E252Q-I272K,A131E-V221T-E252Q-I272K, R117E-A131E-I204Y-G209A-V221T-R229C,R117E-A131E-R229C, R117E-V221T-R229C,Y127D-A131E-I204Y-V221T-E252Q-I272K, Y127D-G209A-V221T-R229C,R117E-G209A-V221T-E252Q, A131E-I204Y-G209A-V221T-E252Q,R117E-Y127D-A131E-I204Y-A217Y-V221T-R229C-E252Q, R117E-A131E-I272K,R117E-A131E-G209A-V221T, Y127D-A131E-A217Y-V221T-E252Q,A131E-I204Y-V221T-I272K, Y127D-G209A-V221T-E252Q-I272K,A131E-I204Y-V221T, Y127D-A131E-I204Y-E252Q,Y127D-A131E-I204Y-G209A-V221T-I272K, Y127D-A131E-G209A-E252Q-I272K,Y127D-A131E-I204Y-G209A-V221T-R229C-E252Q,Y127D-V221T-R229C-E252Q-I272K, A131E-V221T-R229C-E252Q,Y127D-I204Y-V221T-E252Q, R117E-Y127D-A131E-A217Y-V221T-E252Q-I272K,G209A-V221T-R229C-E252Q, R117E-I204Y-V221T,Y127D-I204Y-V221T-R229C-I272K, R117E-V221T-E252Q-I272K,R117E-A131E-V221T, I204Y-V221T-E252Q-I272K,R117E-Y127D-A131E-I204Y-A217Y-V221T-E252Q-I272K,A131E-G209A-V221T-R229C-I272K,Y127D-A131E-I204Y-V221T-R229C-E252Q-I272K, R117E-V221T-I272K,Y127D-A131E-I204Y-I272K, A131E-E252Q,R117E-A131E-I204Y-G209A-V221T-E252Q-I272K,Y127D-A131E-G209A-R229C-E252Q, A131E-G209A-V221T-R229C,R117E-A131E-I204Y-G209A-V221T-I272K, R117E-A131E-I204Y-E252Q,R117E-A131E-I204Y-V221T-R229C-E252Q, A131E-G209A-V221T-I272K,Y127D-E252Q, R117E-A131E-I204Y-G209A-V221T-R229C-E252Q-I272K,R117E-V221T-R229C-E252Q, Y127D-A131E-I204Y-G209A-R229C-E252Q-I272K,R117E-A131E-I204Y-I272K, R117E-A131E-I204Y-V221T-R229C-I272K,R117E-A131E-A217Y-V221T-E252Q, I204Y-G209A-V221T-E252Q-I272K,R117E-Y127D-I204Y-E252Q, I204Y-G209A-V221T,R117E-Y127D-A131E-G209A-E252Q-I272K, R117E-A131E-R229C-E252Q,Y127D-A131E-I204Y-G209A-V221T-R229C-I272K,Y127D-A131E-I204Y-G209A-V221T-R229C,Y127D-A131E-G209A-R229C-E252Q-I272K,A131E-I204Y-G209A-V221T-E252Q-I272K, Y127D-A131E-I204Y-E252Q-I272K,Y127D-A131E-I204Y, R117E-I204Y-G209A-V221T-E252Q,R117E-A131E-G209A-V221T-R229C-E252Q, A131E-I204Y-G209A-V221T-R229C,R117E-V221T-R229C-I272K, Y127D-I204Y-G209A-E252Q-I272K,G209A-V221T-R229C-E252Q-I272K, Y127D-I204Y-V221T-R229C,Y127D-I204Y-G209A-V221T-I272K, R117E-A131E-V221T-I272K,Y127D-I204Y-G209A-V221T-E252Q, L203S-E252Q, L203S-E252Q-I272P,M119V-L203S-E252Q, L203S-S227V-E252Q, M119V-L203S-S227V-E252Q-I272P,M119V-L203S-S227V-E252Q-I272V, R117Q-M119V-L203S-S227V-E252Q-I272P,R117Q-M119V-L203S-S227V-E252Q-I272V,T142V-K185Q-L203S-S227V-E252Q-I272V-K286Q,M119V-K185Q-L203S-S227V-E252Q-I272V-K286Q,M119V-T142V-L203S-S227V-E252Q-I272V-K286Q,M119V-T142V-K185Q-S227V-E252Q-I272V-K286Q,M119V-T142V-K185Q-L203S-S227V-I272V-K286Q,M119V-T142V-K185Q-L203S-S227V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227D-R229V-E252Q,M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q-I272V,R117Q-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q-I272V,R117Q-M119V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-I204Y-G206Q-S227V-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-S227V-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227D-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q-I272P,A131E-L203K-G206Q-R229V-E252Q-I272V,A131E-L203T-G206Q-R229V-E252Q-I272V, A131E-L203K-R229V-E252Q-I272V,A131E-L203K-G206Q-E252Q-I272V, L203T-G206Q-R229V-E252Q-I272V,A131E-L203T-G206Q-R229V-I272V,S095Q-A131E-L203K-G206Q-R229V-K247R-E252Q-I272V,S095Q-L203K-G206Q-R229V-K247R-E252Q-I272V,S095Q-A131E-L203T-G206Q-R229V-K247R-E252Q-I272V,S095Q-A131E-G206Q-R229V-K247R-E252Q-I272V,S095Q-A131E-L203K-R229V-K247R-E252Q-I272V,S095Q-A131E-L203K-G206Q-K247R-E252Q-I272V,S095Q-A131E-L203K-G206Q-R229V-E252Q-I272V,S095Q-A131E-L203K-G206Q-R229V-K247R-I272V,S095Q-A131E-L203K-G206Q-R229V-K247R-E252Q,A131E-L203S-G206Q-R229V-I272P, L203S-G206Q-R229V-I272P, A131E-L203S-R229V-I272P, A131E-L203S-G206Q-I272P, A131E-L203S-G206Q-R229V,R117Q-L203T-A224S-E252Q,R117Q-A131E-L203S-I204Y-S227V-R229Q-E252Q-I272P,A131E-L203S-I204Y-S227V-R229Q-E252Q-I272P,R117Q-L203S-I204Y-S227V-R229Q-E252Q-I272P,R117Q-A131E-I204Y-S227V-R229Q-E252Q-I272P,R117Q-A131E-L203S-S227V-R229Q-E252Q-I272P,R117Q-A131E-L203S-I204Y-R229Q-E252Q-I272P,R117Q-A131E-L203S-I204Y-S227V-R229Q-I272P,R117Q-A131E-L203S-I204Y-S227V-R229Q-E252Q,A131E-L203S-G206Q-A217Y-R229V-I272P,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-A217Y-S227D-R229V-E252Q,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-A217Y-S227D-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-A217Y-S227V-R229V-E252Q-I272P,R117Q-M119V-R120V-A131E-L203T-I204Y-A205W-G206Q-S227D-R229V-E252Q,R117Q-M119V-R120V-A131E-L203T-I204Y-A205W-G206Q-S227D-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-A205W-G206Q-S227V-R229V-E252Q-I272P,R117E-Y127D-A131E-I204Y-A205W-A217Y-V221T-R229C-E252Q,R117G-Y127D-A131E-I204Y-A217Y-V221T-R229Q-E252Q,R117G-Y127D-A131E-I204Y-A205W-A217Y-V221T-R229Q-E252Q,R117G-Y127D-A131E-A217Y-V221T-E252Q-I272K,R117G-Y127D-A131E-A205W-A217Y-V221T-E252Q-I272K,R117G-A131E-I204Y-A217Y-V221T-R229Q-E252Q,R117G-A131E-I204Y-A205W-A217Y-V221T-R229Q-E252Q,R117G-A131E-I204Y-G209A-A217Y-V221T-R229Q-E252Q-I272K,R117G-A131E-I204Y-A205W-G209A-A217Y-V221T-R229Q-E252Q-I272K,R117E-A131E-A205W-A217Y-V221T-E252Q,Y127D-A131E-I204Y-A205W-G209A-A217Y-V221T-R229Q, R117E-A131E-A217Y,A131E-A217Y, L203R-A217Y, L203R-G209D-A217Y, G209D-A217Y,Y022F-R117Q-L203R-G209D-A217Y,Y022F-A159D-L203R-G206C-K208Q-G209D-A217Y-I272P,R117E-A131E-I204Y-G209A-A217Y, R117E-A131E-I204Y-G209A-A217Y-V221T,R117E-A131E-I204Y-G209A-A217Y-V221T-R229C-E252Q-I272K,R117E-A131E-F143L-I204Y-G209A-A217Y-V221T-R229C-E252Q-I272K,Y022F-L203R-G206C-K208Q-G209D-A217Y, Y022F-R117E-A131E-L203R-G209D,Y022F-R117E-A131E-L203R-G209D-A217Y, R117E-A131E-L203R-G209D,R117E-A131E-L203R-G209D-A217Y, R117E-A131E-L203R-I204Y-G209A-V221T,R117E-A131E-L203R-I204Y-G209A-A217Y-V221T, A217Y-E252Q,R117E-A131E-I204Y-G209A-A217Y-E252Q,Y022F-R053Q-R117Q-F143L-L203R-G206C-K208Q-R229V-E252Q,Y022F-R117D-F143L-I272P, Y022F-R117E-A131E-A217Y-V221T,Y022F-R117E-A131E-F143L-A217Y-V221T, A131E-L203R-A217Y-I272P,A131E-F143L-L203R-A217Y-I272P, Y022F-R053Q-F143L-R229V-E252Q-I272P,R117E-A131E-A217Y-V221T-E252Q-I272K,R117E-Y127D-A131E-A217Y-E252Q-I272K,R117E-Y127D-A131E-A217Y-V221T-E252Q,R117E-Y127D-A131E-A217Y-V221T-I272K,R117E-Y127D-A131E-I204Y-G209A-A217Y-V221T-E252Q-I272K,R117E-Y127D-A131E-F143L-I204Y-G209A-A217Y-V221T-R229C-E252Q-I272K,Y127D-A217Y, R117E-A131E-A217Y-V221T,Y022F-A159D-L203R-G209D-A217Y-V221T-I272K,Y022F-P125E-A131E-I179K-L203R-G209D-A217Y-V221T-R229C-I272K,A131E-A217Y-V221T, A131E-G209D-V221T-I272K,A131E-L203R-G209D-A217Y-V221T,A131E-L203R-G209D-A217Y-V221T-R229C-I272K,R117E-Y127D-L203S-A217Y-E252Q, Y022F-A217Y-E252Q,Y022F-L203R-G206C-G209D-A217Y-E252Q,R117Q-A131E-L203S-G209D-A217Y-E252Q, R117Q-Y127D-A217Y-E252Q,A131D-R229V-E252Q-I272P, Y022F-A131D-A217Y-R229V-E252Q-I272P,Y022F-A131E-R229V-E252Q-I272P, Y022F-A131D-R229V-E252Q-I272K,Y022F-A131E-R229V-E252Q-I272K,R117E-Y127D-A131D-A217Y-V221T-E252Q-I272P,R117E-Y127D-A131D-A217Y-V221T-R229C-E252Q-I272P,R117E-Y127D-A131D-A217Y-V221T-R229V-E252Q-I272P,Y127D-A131E-A217Y-V221T-R229V-E252Q-I272K,Y127D-A131E-A217Y-V221T-R229C-E252Q-I272K,Y127D-A131E-A159D-A217Y-V221T-R229C-E252Q-I272K, L203R-R229V-E252Q,L203R-G209D-A217Y-E252Q, L203R-G209A-A217Y-E252Q, L203R-A217Y-R229V,L203R-G209D-A217Y-R229Q-E252Q, L203R-G209D-A217Y-R229V-E252Q,A159D-L203R-G209D-A217Y-E252Q, A159D-L203R-G209D-A217Y,A159D-L203R-G209D-A217Y-I272K, Y022F-L203R-G209D-A217Y,Y022F-L203R-G209D-A217Y-E252Q,Y127D-A131E-I204Y-A217Y-V221T-R229C-E252Q-I272K,Y127D-A131E-L203R-A217Y-V221T-R229C-E252Q-I272K,L203S-G209D-A217Y-R229A, L203N-A217Y-A224S-R229A,R117Q-L203S-A217Y-A224S-S227D-R229S, R117Q-K121S-L203R-R229C,R117Q-A131E-L203R, L203C-A217Y-A224S-R229P,R117Q-K121S-L203C-G206E-A217Y-S227D-R229S,A131E-L203R-G209D-A224S-R229P, R120Q-A131E-L203R,R117Q-L203R-A217Y-A224S-S227D-R229A, A217Y-A224S-R229A,L203R-S227D-R229Q, A131E-L203R-G209D-R229S, K121S-L203R,R117S-L203C-G206E-A217Y-R229Q, R117E-L203R-G209D-A224S-R229S,R120Q-L203C-G209D-R229S, L203R-K208E-G209D-A224S,R117S-R120Q-K121S-L203R-A217Y-A224S-R229C, L203R-A224S,R117Q-A131E-L203C-A217Y-S227D-R229P, R117S-L203S-A224S,Y127E-L203S-A217Y-R229C, R117Q-L203R-G206E-G209D-A217Y-S227D,R117S-K121S-L203R-R229P, R117S-R120Q-A131E, R117E-G209D,R117S-R120Q-Y127E-L203R-G206E-A217Y, L203R-A217Y-A224S-R229Q,R120Q-G206E-G209D-A217Y-A224S, R117E-R120Q-L203R-G209D-A217Y-A224S,A131E-G209D-A217Y-A224S-R229A, G209D-A224S,R117S-Y127E-A217Y-A224S-S227D-R229C, K121E-L203N-A217Y-R229A,Y127E-A217Y-A224S-R229S, R117S-A131E-L203S-R229S, L203R-K208E-R229A,R117Q-A131K-L203R-A224S, L203R-R229A, R120E-L203R-R229P, R120Q-A217Y,R120Q-L203N-A217Y-A224S-R229S, L203R-A217Y-A224S-R229A,L203S-K208E-G209D-A217Y-A224S-S227D-R229S, R117S-G209D-A217Y,L203S-G209D-S227D, A131E-L203R-A224S-R229A, L203R-A224S-R229P,Y127E-L203R-A217Y-R229C, R117Q-L203R-R229S, K121S-L203R-A217Y-A224S,R117S-R120Q-L203R-R229Q, R117S-R120Q-L203R, R117S-L203R-R229A,R117S-L203R-A224S, R117Q-S227D-R229A, L203C-G209D-A217Y-S227D,R120Q-L203R-A217Y, R117S-L203R-A224S-R229C, K121S-L203C-R229A,K121E-L203N-A217Y-A224S-S227D-R229P, K121E-L203R-A217Y,A131E-G209D-A217Y, L203N-A224S-S227D-R229C, K121E-L203R-R229C,R117Q-L203R-G209D-R229A, R117Q-A224S-R229C, R117S-L203R-A224S-R229A,R117Q-K121S-L203R-S227D-R229P, R117S-Y127E-L203S-A224S-S227D,R117S-L203R-R229Q, R117Q-L203R, A131E-L203R-A224S-R229S,K121S-A131E-L203R-A224S, R117S-K121S-L203C-G206E-A217Y-A224S-S227D,R117Q-G209D, R120Q-A131E-L203R-R229C,R117Q-R120Q-Y127E-L203S-A217Y-A224S, R117Q-R120Q-L203R-A217Y-A224S,R117Q-R120Q-L203R-A217Y, R120Q-L203R-A217Y-A224S-S227D-R229S,R117Q-R120E-L203R-R229Q, R117Q-R120E-L203R-A217Y-A224S,A131E-L203R-A224S, R117S-A131E-L203N-S227D, L203S-R229C,L203S-A224S-S227D-R229A, L203R-G209D-R229C, L203R-A217Y-R229A,L203R-G206E-A217Y-S227D-R229P, L203R-A217Y-R229S,R117Q-K121E-L203R-A224S-S227D,R117S-K121S-L203C-G206E-A217Y-A224S-S227D-R229S,R117S-K121S-L203N-A217Y-R229Q, R117Q-L203S-R229C,R117Q-L203R-G206E-A224S-R229S, R117Q-R120E-L203R,R120E-G206E-A217Y-R229Q, R117S-L203S-K208E,R120Q-K121E-L203C-A217Y-R229A, A131E-L203R-A217Y-A224S-R229A,R120E-L203R-G209D-R229Q, A131E-L203R-S227D-R229P,L203R-G209D-A217Y-A224S-R229S, L203N-G206E-A217Y,R120Q-L203R-G206E-A217Y-R229Q,R117Q-K121S-L203R-G206E-A217Y-S227D-R229Q, R120E-L203R-S227D-R229A,R117S-L203R-A224S-R229S, R120Q-L203R-A224S-R229C, L203R-R229S,L203R-R229P, R117Q-K121E-L203R-R229P, R117E-R120Q-L203R-A224S,R120Q-A131E-L203C-A217Y-A224S-R229S,R117Q-R120Q-L203S-A217Y-A224S-R229C,R117Q-A131E-L203R-G206E-A217Y-A224S-S227D-R229A,R120Q-K121E-L203C-A217Y-A224S, A131E-L203N-A217Y-A224S-S227D,R117E-L203R-G209D, R120Q-L203C-G206E, L203R-G209D-S227D,R117S-L203C-G206E-A217Y-A224S, R117E-K121E-L203R,R117E-A131E-L203N-G209D-A217Y, L203C-S227D, L203S-A217Y-R229Q,R117E-A217Y-S227D, R117Q-K121S-L203R-R229Q, L203R-K208E-G209D,R117S-L203S-G206E-G209D, R117S-K121E-L203R-A224S-R229P,L203C-A217Y-S227D-R229C, R117Q-K121S-L203R,R117Q-R120Q-Y127E-L203R-A217Y-A224S, K121E-L203S-A217Y-R229P,R120Q-L203R-G206E-A217Y-A224S, I154V-K170E-A217Y, V039I-A217Y,A217Y-V265A, Y026C-K208E-A217Y, E067G-I204V-A217Y, K170E-A217Y,K134E-A217Y, A217Y-V235A, F060L-A217Y-E252G, 1064T-A217Y-V261A,111T-A217Y, M166T-A217Y, A093S-R120S-A217Y, K170S-A217Y,S141N-A217Y-A218D-E252D, S141N-A217Y-E252G, I019L-N149I-F196L-A217Y,H011R-1064V-K134M-E163G-A217Y-E252V andT004K-A070V-K071E-R117Q-A131T-F143L-N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229H-N280K.7. The lipolytic enzyme variant or active fragment thereof of any ofembodiments 1-6, wherein the variant or active fragment has lipolyticactivity.8. The lipolytic enzyme variant or active fragment thereof of anypreceding embodiments, wherein said lipolytic enzyme variant or activefragment thereof has an improved performance relative to the parentlipolytic enzyme, wherein the improved performance is selected from thegroup consisting of an improved wash performance, an increased detergentstability, an increased thermostability, and any one combinationthereof.9. The lipolytic enzyme variant or active fragment thereof of any ofembodiments 1-7, wherein the variant or active fragment has a detergentstability that is greater that the detergent stability of the parentlipolytic enzyme.10. The lipolytic enzyme variant or active fragment thereof of any ofembodiments 1-7, wherein the variant or active fragment has a washperformance index (PI(wash)) relative to the parent lipolytic enzymethat is greater than 1.0.11. A composition comprising at least one lipolytic enzyme variant orfragment according to embodiment 1.12. The composition according to embodiment 11, wherein said compositionis a detergent composition.13. The composition according to embodiment 12, wherein said detergentcomposition is selected from a laundry detergent, a fabric softeningdetergent, a dishwashing detergent, and a hard-surface cleaningdetergent.14. The composition of embodiment 11, wherein said composition furthercomprises one or more calcium ions and/or zinc ions; one or more enzymestabilizers; from about 0.001% to about 1.0 weight % of said lipolyticenzyme variant(s); one or more bleaching agents; one or more adjunctmaterials; and/or one or more additional enzymes or enzyme derivativesselected from the group consisting of acyl transferases, alpha-amylases,beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases,beta-galactosidases, carrageenases, catalases, cellobiohydrolases,cellulases, chondroitinases, cutinases, DNase or nuclease, endo-beta-1,4-glucanases, endo-beta-mannanases, esterases, exo-mannanases,galactanases, glucoamylases, hemicellulases, hyaluronidases,keratinases, laccases, lactases, ligninases, lipases, lipoxygenases,lysozymes, mannanases, oxidases, pectate lyases, pectin acetylesterases, pectinases, pentosanases, perhydrolases, peroxidases,phenoloxidases, phosphatases, phospholipases, phytases,polygalacturonases, proteases, pullulanases, reductases,rhamnogalacturonases, beta-glucanases, tannases, transglutaminases,xylan acetyl-esterases, xylanases, xyloglucanases, xylosidases,metalloproteases, nucleases, additional serine proteases, andcombinations thereof.15. The composition of embodiment 11, wherein said composition is agranular, powder, solid, bar, liquid, tablet, gel, paste or unit dosecomposition.16. A DNA sequence encoding the lipolytic enzyme variant or activefragment of embodiment 1.17. An expression vector or cassette comprising the DNA sequence ofembodiment 16.18. The expression vector or cassette of embodiment 17, wherein the DNAsequence is operably linked to a promoter.19. A recombinant host cell comprising the DNA sequence of embodiment16.20. Use of the lipolytic enzyme variant or fragment of embodiment 1 forhydrolyzing a lipolytic enzyme substrate.21. A method for cleaning a surface comprising contacting the surfacewith a composition comprising at least one lipolytic enzyme variant ofembodiment 1, and optionally a surfactant.22. A method of producing a lipolytic enzyme variant comprisingculturing the host cell comprising a DNA sequence encoding the lipolyticenzyme variant or active fragment of embodiment 1 in a culture mediumunder conditions conducive for the production of the lipolytic enzymevariant and recovering the lipolytic enzyme variant from said culturemedium.23. A lipolytic enzyme variant or an active fragment thereof comprisingat least two amino acid modifications to a parent lipolytic enzyme,wherein the variant or active fragment thereof comprises amino acidmodifications selected from the group consisting of L203R-G206C-I272P,P125E-L203R-K208Q-G209D, L203R-E252Q-I272P,Y022F-R117Q-A131T-F143L-L203R-G209D-R229Q, L203S-K208Q-G209D-R229T,R117Q-L203R-G209D-R229C, L203R-G209A-I272P, A131D-L203S-R229T-I272P,R117E-A131E-I204Y-E252Q-I272K, Y127D-A131E-A217Y-V221T-E252Q,L203R-I204Y-I272P, Y127D-I204Y-V221T-R229C-I272K,Y127D-A131E-I204Y-G209A-V221T-I272K,R117E-A131E-I204Y-G209A-V221T-E252Q-I272K,Y127D-V221T-R229C-E252Q-I272K, L203S-K208Q-R229T-I272P,Y127D-L203R-I272P, Y127D-I204Y-V221T-R229C,R117E-A131E-I204Y-G209A-V221T-I272K,R117E-A131E-I204Y-G209A-V221T-R229C, R117E-A131E-I204Y-I272K,A131E-L203R-K208Q, R117Q-L203R-K208Q, L203R-R229T-A262M,R117E-V221T-R229C-E252Q, A131E-G209A-V221T-R229C,G209A-V221T-R229C-E252Q, R117E-A131E-G209A-V221T-R229C-E252Q,R117E-A131E-I204Y-V221T-R229C-E252Q, R117E-I204Y-G209A-V221T-E252Q,A131E-L203R-G209D,T004K-A070V-K071E-R117Q-A131T-F143L-N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229H-N280K,Y127D-A131E-I204Y-G209A-R229C-E252Q-I272K, L203R-R229T-E252Q,Y127D-A131E-I204Y-V221T-R229C-E252Q-I272K, L203R-G209D-I272P,L203R-G209D-R229Q, L203R-K208Q, L203R-K208Q-E252Q, R117Q-L203R-R229T,Y022F-R117Q-R229V-I272P, R117E-A131E-I204Y-V221T-R229C-I272K,A131D-L203R-E252Q, L203R-K208Q-R229T-I272P, R117Q-A131D-E252Q-I272P,R117E-A131E-I204Y-G209A-V221T-R229C-E252Q-I272K,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-E252Q-I272V,Y022F-R053Q-R117Q-L203R-R229V-E252Q, R117E-A131E-R229C,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q-I272P,Y127D-I204Y-G209A-E252Q-I272K, L203R-G209D, L203R-I272P,L203R-K208Q-A262M, R117E-A131E-R229C-E252Q,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-A217Y-S227D-R229V-E252Q-I272V,L203R-R229T-I272P,R117G-A131E-I204Y-A205W-G209A-A217Y-V221T-R229Q-E252Q-I272K,R117Q-A131E-L203S-I204Y-S227V-R229Q-E252Q-I272P,Y022F-R053Q-R117Q-E252Q-I272P, Y022F-L203R-R229Q,Y022F-R117Q-E252Q-V261L, L203R-G209D-E252Q, Y127D-G209A-V221T-R229C,L203R-R229Q, R117Q-L203R-I272P, L203R-K208Q-G209D-R229T,R117S-L203R-A224S-R229S, L203R-G206C-G209D, R117E-A131E-I204Y-E252Q,Y127D-A131E-I204Y-G209A-V221T-R229C, L203N-A217Y-A224S-R229A,L203R-I204Y, Y022F-L203R-R229T, Y127D-A131E-G209A-R229C-E252Q-I272K,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-A217Y-S227V-R229V-E252Q-I272P,Y022F-R117Q-G209D-R229V-E252Q-I272P,R117Q-M119V-R120V-A131E-L203T-I204Y-A205W-G206Q-S227V-R229V-E252Q-I272P,R117S-L203C-G206E-A217Y-R229Q, Y022F-L203R-I272P andR117Q-L203S-A217Y-A224S-S227D-R229S, wherein the amino acid positions ofthe variant are numbered by correspondence with the amino acid sequenceof SEQ ID NO: 1, wherein the lipolytic enzyme variant has at least 60%sequence identity to the amino acid sequence SEQ ID NO: 1, and whereinsaid lipolytic enzyme variant or active fragment thereof has an improvedwash performance relative to the wash performance of said parentlipolytic enzyme.24. The lipolytic enzyme variant or active fragment thereof ofembodiment 23, wherein the variant or active fragment has a detergentstability that is greater that the detergent stability of the parentlipolytic enzyme set forth in SEQ ID NO: 1.25. The lipolytic enzyme variant or active fragment thereof ofembodiment 23, wherein the variant or active fragment has a washperformance index (PI(wash)) relative to the parent lipolytic enzyme setforth in SEQ ID NO: 1 that is greater than 1.0.

EXAMPLES

In the following Examples, unless otherwise stated, parts andpercentages are by weight and degrees are Celsius. It should beunderstood that these Examples, while indicating embodiments of thedisclosure, are given by way of illustration only. From the abovediscussion and these Examples, one skilled in the art can make variouschanges and modifications of the disclosure to adapt it to varioususages and conditions. Such modifications are also intended to fallwithin the scope of the appended claims.

Example 1 Lipase Variants

Synthetic genes encoding a parent lipase amino acid sequence derivedfrom Proteus vulgaris, set forth in SEQ ID NO: 1, and lipase variantsthereof were designed for expression in Bacillus subtilis. These geneswere cloned and expressed in Bacillus subtilis and their expression wassubsequently evaluated.

Lipase titers (enzyme concentration) were determined based on antibodyassociation rates measured by Biolayer Interferometry using the OctetHTX instrument (ForteBio). Biolayer interferometry measures changes inthe absorption of visible light at the surface of an optical probecaused by the accumulation of protein biolayer. To measure lipaseconcentrations in crude Bacillus subtilis supernatants we obtainedstreptavidin-coated biosensors (ForteBio), and functionalized them witha biotin-labeled polyclonal antibody that recognize lipase variants ofSEQ ID NO:1. Polyclonal antibodies were isolated from serum extractedfrom rabbits inoculated with purified native protein from a lipasevariant of SEQ ID NO: 1, referred to asA029R_I064V_S095N_S227T_R267P_I272V, (SEQ ID NO: 2). Lipase reactivesensors prepared in this format facilitate the specific accumulation ofprotein from SEQ ID NO:1 and related variants at the sensor surface whensubmerged in solutions containing known and unknown concentrations oflipase. Association rates from a SEQ ID NO:1 dilution series ofpre-determined concentrations were fit to a four-parameter linearequation using ForteBio Data Analysis 10.0 software (ForteBio) and theequations were used to calculate titer values from the association ratesmeasured in supernatants containing unknown amounts of SEQ ID NO: 1lipase and variants thereof.

Bacillus subtilis fermentate UFC was produced by standard methods andwas filtered through a 0.2 um filter and dialyzed into 10 mM PotassiumPhosphate, 0.01% Triton X-100, pH 7.0. Sample was purified using CationExchange Chromatography with an Akta Pure (GE Healthcare Life Sciences).In detail, dialyzed material was loaded onto a HiPrep 16/60 SP HP at a 3mL/min flowrate, and eluted using a 30% gradient over 7 column volumeswith 10 mM Potassium Phosphate, 1M NaC pH 7.0. Elution was collected in4 mL fractions and pooled based on SDS-PAGE identification of a bandcomprising of the correct size and 4-methylumbelliferone (4-MU)caprylate hydrolysis activity. Pooled fractions were concentrated andbuffer exchanged into 25 mM HEPES, 50 mM KCl, 2 mM CaCl₂), 5% glycerol,pH 7.0.

For purified lipase samples, microvolume protein concentrationdetermination, in mg/mL, was obtained by using a Thermo ScientificNanoDrop 2000 Spectrophotometer. Specifically, a blank measurement wasestablished by using a 2 μL aliquot of storage buffer (25 mM HEPES, 50mM KCl, 2 mM CaCl₂), 5% glycerol, pH 7.0). Then, an aliquot of 2 μLlipase sample was used to measure the absorbance at 280 nm. Atheoretical molar extinction coefficient (ε=21,170 L·mol-1·cm-1) and amolecular weight of 31,280 Da were used to convert the Absorbance tomg/mL of protein by the NanoDrop Software. The measurement was repeatedthree to five times and the average value was reported.

Example 2 General Methods

The following assays are standard assays used in the examples describedbelow. Occasionally specific protocols call for deviations from thesestandard assays. In those cases, deviations from these standard assayprotocols below are identified in the examples.

A. Wash Performance Index, PI(wash)

The wash performance index, PI(wash), of an enzyme compares theperformance of the variant with a parent or reference polypeptide at thesame protein concentration.

A PI(wash) that is greater than 1 (PI>1) indicates improved washperformance by a variant as compared to the parent or referencepolypeptide (e.g., SEQ ID NO:1), while a PI of 1 (PI=1) identifies avariant that performs the same in washing as the parent or referencepolypeptide, and a PI that is less than 1 (PI<1) identifies a variantthat does not perform as well in wash performance as the parent orreference polypeptide.

The wash performance index (PI(wash)) of a lipase relative to areference lipase was calculated as:

PI(wash)=((% SRI(variant)−% SRI(no enzyme))/((% SRI(lipase ref.)−%SRI(no enzyme)).

B. Hydrolysis of 4-Methylumbelliferone Caprylate Assay

The Lipase variants can be assayed for lipase activity on a4-Methylumbelliferone (4-MU) caprylate substrate (octanoate) (Chem-ImpexInternational, CAS20671-66-3). A reaction emulsion with 4-MU caprylateis prepared using 4-MU caprylate pre-suspended in ethanol (100%) andbuffer. The 4-MU caprylate/buffer suspensions are mixed and transferredto a 96-well microtiter plate (MTP) containing the enzyme sample, in atotal volume of 100 μL. Dilution of the enzyme samples and theirtransfer volumes are adjusted to keep the reaction within a linearrange. The release of 4-methylumbelliferone due to hydrolysis wasmonitored by measuring the fluorescence with the excitation at 380 nmand the emission at 450 nm for a period of 3 to 5 minutes in 15-18second intervals with a SpectraMax (Molecular Devices) corrected usingblank values (no enzyme). The enzyme hydrolysis rate is calculated bydividing the fluorescence units measured over time.

C. Wash Performance Assays Miniswatch Assay

The miniswatch assay was performed as follows. Detergent solutions wereprepared by weighing out the appropriate mass of heat-treated detergent(Heat-inactivated commercial HDL laundry detergent, 8 h at 80° C.). Avolume of 3:1 Ca²⁺:Mg²⁺ Hardness stock solution was added to thedetergent so that the final working solution contained 250 ppm of 3:1Ca²⁺:Mg²⁺, then water was added to achieve 95% the final working volume.Enzyme solutions were prepared as a 20× solution in 200 mM HEPES pH 8.0.When the 20× Enzyme solution was added to the 95% detergent solution thefinal working cleaning solution contained 1× enzyme, 10 mM HEPES pH 8.0,250 ppm hardness and a manufacturer-recommended concentration ofdetergent. CS-61 swatches, which are pre-stained cotton swatches stainedwith beef fat and a red dye (Center for Testmaterial, CFT, TheNetherlands) were used in a 24-well plate format. Swatches were preparedby cutting 12-mm square swatches from larger stained swatch materialobtained from Center for Testmaterial (Center for Testmaterial, CFT, TheNetherlands). One square of CS-61 and 1.9 mL of the 95% detergentdilution was added to each well, and 100 μL of the 20× enzyme dilutionwas added to each well containing detergent. Plates were sealed andincubated in a shaker incubator at 400 rpm for 30 min at 16° C. Shakingconditions were optimized using an Infors shaker with a 3-mm throw.After the incubation period, swatches were removed from the wells usingtweezers and rinsed under cold running tap water to remove excessdetergent and enzyme. Rinsed swatches were then placed on an absorbentpaper towel and allowed to dry completely protected from light. Colormeasurements of the dried swatches were performed using a hand-heldreflectometer. Each individual piece of washed swatch material wasplaced on a black paper sheet and interrogated with the reflectometer.L*a*b* values were recorded and % Soil Removal Index (% SRI) wascalculated using unwashed technical stain for before wash, washedtechnical stain for after wash and a white reflectometer calibrationtarget as a reference measure of white.

CIE L*a*b* measurements of technical stains were made using a MinoltaCR-400 Reflectometer with 8 mm aperture. % Soil Removal Index (% SRI)was calculated using the following equation:

${\%{SRI}} = {\frac{{\Delta E}\left( {{After} - {B{efore}{Wash}}} \right)}{\Delta{E\left( {{White} - {{Before}{Wash}}} \right)}} = {\frac{\sqrt{\left( {L_{f} - L_{i}} \right)^{2} + \left( {a_{f} - a_{i}} \right)^{2} + \left( {b_{f} - b_{i}} \right)^{2}}}{\sqrt{\left( {L_{w} - L_{i}} \right)^{2} + \left( {a_{w} - a_{i}} \right)^{2} + \left( {b_{w} - b_{i}} \right)^{2}}} \times 100}}$

where L_(f) is L* for after wash, L_(i) is L* for before wash, and L_(w)is L* for white, where a_(f) is a* for after wash, a_(i) is a* forbefore wash, and a_(w) is a* for white, and where b_(f) is b* for afterwash, b_(i) is b* for before wash, and b_(w) is b* for white, and whereΔE is defined as color difference as defined by L*a*b*.

Miniswatch assays used commercial laundry detergents. For North American(NA) and Western European (WE) heavy duty liquid laundry (HDL)detergents, heat inactivation of the enzymes present incommercially-available detergents was performed by placing pre-weighedliquid detergent (in a sealed glass bottle) in a water bath at 80° C.for 8 hours. For testing of enzyme activity in heat-inactivateddetergents, wash solutions of detergents were made from the heatinactivated HDL detergent stocks. Appropriate amounts of water hardness(e.g., 14.6 gpg or 17.5 gpg) and buffer were added to the wash solutionsto match the desired conditions. The wash solutions were mixed bystirring prior to use.

In one aspect, a Persil Color Gel detergent (HDL detergent, WE region)was used wherein the laundry washing conditions were at a dose of 8.0g/L detergent, 5 mM HEPES buffer (pH 8.0), 17.5 gpg of 3:1 Ca²⁺:Mg²⁺,and temperature at 16° C.

D. Detergent Stability Assays

Stability in HDL detergent was assessed by incubating lipase samples inHDL formulations at elevated temperatures and comparing the activitymeasured following the stress treatment to an unstressed paired sample.After the stress period, samples were frozen until assayed. Theunstressed paired sample was mixed with the HDL formulation butimmediately frozen.

In one aspect, lipase samples were incubated in a model HDL formulation.The model HDL comprised the following ingredients: 3.1% lauryl alcoholethoxylate (6EO), 9% sodium lauryl ether sulphate, 3% sodium citrate,0.8% sorbitol, 0.8% glycerol, 0.5% triethanolamine, 1% ethanol(absolute); 7.6% linear alkylbenzenesulfonate (LAS), 3.0% potassiumcocoate, 2.5% propylene glycol, 0.01% 2-methyl-4-isothiazolin-3-one,5.8% 4M NaOH and 62.9% demineralized water.

In another aspect, lipase samples were incubated in a heat-inactivatedcommercial HDL formulation. For North American (NA) and Western European(WE) heavy duty liquid laundry (HDL) detergents, heat inactivation ofthe enzymes present in commercially-available detergents was performedby placing pre-weighed liquid detergent (in a sealed glass bottle) in awater bath at 80° C. for 8 hours. In one aspect, a Persil Color Geldetergent (HDL detergent, WE region) was used.

After stress incubations, all samples were equilibrated to roomtemperature and diluted 10- to 15-fold in 10 mM HEPES pH 8.0. Dilutedsamples were mixed by pipetting and a 40 μL volume was transferred to anopaque assay plate. A substrate solution was freshly preparedimmediately prior to enzyme activity measurements that contained 2 mM4-methylumbelliferyl-caprylate and buffer. An 80 μL volume of thesubstrate solution was added to the samples and activity was measured bymonitoring the fluorescent emission of liberated methylumbelliferone. Afluorescent plate reader (Spectromax M5, Molecular Devices) was set toan excitation wavelength of 380 nm and an emission wavelength of 450 nmand fluorescent emission levels were recorded for 3 to 5 minutes at 15to 18 second intervals and raw data was recorded as fluorescentunits/second. Background hydrolysis rates were measured in a no-enzymecontrol well containing buffer components but no added lipase and wasused to subtract background hydrolysis from the sample wells. The ratioof stressed vs. unstressed values for paired samples was used tocalculate the % residual activity. Samples that have a higher % residualactivity than the parental strain SEQ ID NO: 1 are more stable than theparental strain.

Example 3 Lipolytic Enzyme Variants with Improved Stability in LiquidDetergents

The stability of lipase variants of the parent lipase of SEQ ID NO:1described herein was determined as described in Example 2 for Stabilityin HDL detergent. Lipase samples were incubated in a 25% finalconcentration of the model detergent for a 2-hour period of heat stressat 45° C. Lipase activity was subsequently measured as described inExample 2. The reaction emulsion to measure activity consisted of: 1.3mM 4-methylumbelliferyl-caprylate, 10 mM HEPES pH8, 100 mM NaCl, 1 mMCaCl₂), 0.2% Triton X100, and 0.2% bovine serum albumin. Activity levelsmeasured for unstressed controls were compared to activity levels afterheat stress. The ratio of stressed vs unstressed values was used tocalculate the % residual activity and compared to the % residualactivity of the parent lipase (SEQ ID NO:1). The % residual activity forthe parent lipase (SEQ ID NO:1) under these conditions was 0%.

Lipase variants are referred to by their positional modificationrelative to the lipase of SEQ ID NO: 1. For example, lipase variant“N146H_N280K” is a variant of SEQ ID NO: 1 that comprises a substitutionat position 146 (N changed to H in variant) and a substitution atposition 280 (N changed to K in variant). Lipase variants with animproved detergent stability compared to the parent lipase of SEQ ID NO:1 are shown in Table 20.

TABLE 20 Lipase variants with Improved Detergent Stability in ModelLiquid Detergent. % Lipase variant (positions modified Residual relativeto parent of SEQ ID NO: 1) Activity SEQ ID NO: 1  0%T004K-A070V-K071E-R117Q-A131T- 75% F143L-N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229H-N280K Y022F-L203R 67% A131D-L203R 44%Y127D-L203R 44% A131E-L203R 42% L203R-I204Y 60% L203R-G206C 30%L203R-K208Q 52% L203R-G209D 55% L203R-G209A 47% L203R-R229Q 41%L203R-E252Q 47% L203R-V261L 49% L203R-A262M 43% L203R-A262C 41%L203R-I272P 55% Y022F-R117D-L203R 82% Y022F-R117Q-L203R 78%Y022F-A131E-L203R 70% Y022F-L203R-G206C 53% Y022F-L203R-K208Q 70%Y022F-L203R-G209D 72% Y022F-L203R-G209A 71% Y022F-L203R-R229Q 62%Y022F-L203R-R229T 62% Y022F-L203R-I272P 69% R117D-A131E-L203R 66%R117D-L203R-E252Q 71% R117Q-A131D-L203R 64% A131D-L203R-G206C 45%A131D-L203R-R229T 35% A131D-L203R-E252Q 58% R117Q-L203R-K208Q 60%Y127D-L203R-K208Q 44% A131E-L203R-K208Q 62% L203R-I204Y-K208Q 78%L203R-K208Q-G206C 49% L203R-K208Q-G209D 75% L203R-K208Q-G209A 68%L203R-K208Q-R229Q 51% L203R-K208Q-R229T 51% L203R-K208Q-E252Q 64%L203R-K208Q-V261L 65% L203R-K208Q-A262M 62% L203R-K208Q-I272P 75%Y127D-L203R-G209D 68% A131E-L203R-G209D 62% L203R-I204Y-G209D 78%L203R-G206C-G209D 52% L203R-G209D-R229Q 51% L203R-G209D-R229T 50%L203R-G209D-E252Q 69% L203R-G209D-I272P 79% R117Q-L203R-R229T 54%Y127D-L203R-R229T 53% A131E-L203R-R229T 50% L203R-I204Y-R229T 69%L203R-G206C-R229T 29% L203R-G209A-R229T 59% L203R-R229T-E252Q 52%L203R-R229T-A262M 53% L203R-R229T-I272P 63% R117Q-L203R-I272P 71%Y127D-L203R-I272P 65% A131E-L203R-I272P 70% L203R-I204Y-I272P 73%L203R-G206C-I272P 56% L203R-G209A-I272P 71% L203R-E252Q-I272P 61%A131D-L203R-G209D-R229T 75% A131D-L203R-R229T-I272P 75%A131D-L203S-R229T-I272P 66% L203R-K208Q-G209D-R229T 70%L203R-K208Q-R229T-I272P 74% L203S-K208Q-G209D-R229T 62%L203S-K208Q-R229T-I272P 63% Y022F-R117Q-A131T-F143L- 79%L203R-G209D-R229Q Y199H-L203R-G209D-A217Y- 78% R229QL203R-G209D-A217Y-R229Q 92% R117Q-L203R-G209D-R229C 64% Y199H-A217Y 60%A217Y-V221T-R229C-E252Q- 62% A262M-I272P E252Q-A262M-I272P 24%Y022F-A159D-L203R-G206C- 90% K208Q-G209D-I272P Y022F-L203R-G206C-K208Q-81% G209D P125E-L203R-K208Q-G209D 75% Y022F-R229C-E252Q-I272P 36%A159D-L203R-G209D 63% T004K-K071E-R117D-A131T-F143L- 87%N146H-A159D-I179K-L203R-G206E- G209D-L212R-R229C-N280KT004K-K071E-R117Q-A131D-F143L- 85% N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229C-N280K A159D-L203R-G209D-R229H 56%A159D-L203R-G209D-R229C 63% A159D-L203R-G209D-R229V 61%A159D-L203R-G206E-G209D 66% Y022F-R117Q-A131D-L203R- 67% R229V-E252QY022F-R053Q-R117Q-E252Q- 72% V261L-I272P Y022F-R117Q-A131D-E252Q 62%Y022F-R117Q-R229V-E252Q- 56% V261L-I272P Y022F-R053Q-R229V-E252Q-I272P74% R117Q-A131D-E252Q-I272P 57% Y022F-R053Q-R117Q-R229V-V261L 57%Y022F-R117Q-G209D-R229V-E252Q-I272P 59% Y022F-A131D-R229V-E252Q-I272P53% Y022F-RO53Q-R117Q-L203R-R229V- 84% E252Q-V261L Y022F-R117Q-R229V 41%Y022F-R117Q-A131D-R229V-E252Q 64% Y022F-R053Q-R117Q-L203R-R229V- 109% E252Q-V261L-I272P Y022F-R053Q-R117Q-L203R-R229V- 90% E252Q-I272PY022F-RO53Q-R117Q-L203R-R229V- 84% E252QY022F-A131D-L203R-R229V-E252Q-I272P 70% Y022F-R053Q-R117Q-E252Q-I272P81% Y022F-R117Q-E252Q-V261L 59% R117Q-A131D-L203R-R229V-E252Q 70%Y022F-R229V-E252Q-I272P 33% Y022F-R117Q-R229V-I272P 62% Y022F-R117D 57%Y022F-K134E-R229Q 42% Y022F-R117D-I272P 82%R117E-A131E-I204Y-E252Q-I272K 71% A131E-V221T-E252Q-I272K 17%R117E-A131E-I204Y-G209A-V221T-R229C 80% R117E-A131E-R229C 60%R117E-V221T-R229C 47% Y127D-A131E-I204Y-V221T-E252Q-I272K 48%Y127D-G209A-V221T-R229C 73% R117E-G209A-V221T-E252Q 42%A131E-I204Y-G209A-V221T-E252Q 36% R117E-Y127D-A131E-I204Y-A217Y- 102% V221T-R229C-E252Q R117E-A131E-I272K 26% R117E-A131E-G209A-V221T 52%Y127D-A131E-A217Y-V221T-E252Q 82% A131E-I204Y-V221T-I272K 27%Y127D-G209A-V221T-E252Q-I272K 21% A131E-I204Y-V221T 15%Y127D-A131E-I204Y-E252Q 37% Y127D-A131E-I204Y-G209A-V221T-I272K 54%Y127D-A131E-G209A-E252Q-I272K 35% Y127D-A131E-I204Y-G209A-V221T- 94%R229C-E252Q Y127D-V221T-R229C-E252Q-I272K 69% A131E-V221T-R229C-E252Q50% Y127D-I204Y-V221T-E252Q 25% R117E-Y127D-A131E-A217Y-V221T- 92%E252Q-I272K G209A-V221T-R229C-E252Q 52% R117E-I204Y-V221T 30%Y127D-I204Y-V221T-R229C-I272K 58% R117E-V221T-E252Q-I272K 39%R117E-A131E-V221T 28% I204Y-V221T-E252Q-I272K 22%R117E-Y127D-A131E-I204Y-A217Y- 90% V221T-E252Q-I272KA131E-G209A-V221T-R229C-I272K 47% Y127D-A131E-I204Y-V221T-R229C- 76%E252Q-I272K R117E-V221T-I272K 17% Y127D-A131E-I204Y-I272K 34%A131E-E252Q 12% R117E-A131E-I204Y-G209A-V221T- 75% E252Q-I272KY127D-A131E-G209A-R229C-E252Q 72% A131E-G209A-V221T-R229C 59%R117E-A131E-I204Y-G209A-V221T-I272K 76% R117E-A131E-I204Y-E252Q 65%R117E-A131E-I204Y-V221T-R229C-E252Q 83% A131E-G209A-V221T-I272K 19%Y127D-E252Q 10% R117E-A131E-I204Y-G209A-V221T- 84% R229C-E252Q-I272KR117E-V221T-R229C-E252Q 65% Y127D-A131E-I204Y-G209A-R229C- 58%E252Q-I272K R117E-A131E-I204Y-I272K 58%R117E-A131E-I204Y-V221T-R229C-I272K 81% R117E-A131E-A217Y-V221T-E252Q93% I204Y-G209A-V221T-E252Q-I272K 34% R117E-Y127D-I204Y-E252Q 60%I204Y-G209A-V221T 13% R117E-Y127D-A131E-G209A-E252Q-I272K 74%R117E-A131E-R229C-E252Q 67% Y127D-A131E-I204Y-G209A-V221T- 80%R229C-I272K Y127D-A131E-I204Y-G209A-V221T-R229C 84%Y127D-A131E-G209A-R229C-E252Q-I272K 59%A131E-I204Y-G209A-V221T-E252Q-I272K 41% Y127D-A131E-I204Y-E252Q-I272K42% Y127D-A131E-I204Y 18% R117E-I204Y-G209A-V221T-E252Q 61%R117E-A131E-G209A-V221T-R229C-E252Q 79% A131E-I204Y-G209A-V221T-R229C44% R117E-V221T-R229C-I272K 52% Y127D-I204Y-G209A-E252Q-I272K 46%G209A-V221T-R229C-E252Q-I272K 22% Y127D-I204Y-V221T-R229C 60%Y127D-I204Y-G209A-V221T-I272K 35% R117E-A131E-V221T-I272K 45%Y127D-I204Y-G209A-V221T-E252Q 31% L203S-E252Q 34% L203S-E252Q-I272P 50%M119V-L203S-E252Q 75% L203S-S227V-E252Q 46%M119V-L203S-S227V-E252Q-I272P 72% M119V-L203S-S227V-E252Q-I272V 55%R117Q-M119V-L203S-S227V-E252Q-I272P 79%R117Q-M119V-L203S-S227V-E252Q-I272V 63% T142V-K185Q-L203S-S227V-E252Q-60% I272V-K286Q M119V-K185Q-L203S-S227V-E252Q- 60% I272V-K286QM119V-T142V-L203S-S227V-E252Q- 53% I272V-K286QM119V-T142V-K185Q-S227V-E252Q- 20% I272V-K286QM119V-T142V-K185Q-L203S-S227V- 34% I272V-K286QM119V-T142V-K185Q-L203S-S227V- 61% E252Q-I272VR117Q-M119V-R120V-A131E-L203T- 61% I204Y-G206Q-S227D-R229V-E252QM119V-R120V-A131E-L203T-I204Y- 77% G206Q-S227V-R229V-E252Q-I272VR117Q-R120V-A131E-L203T-I204Y- 86% G206Q-S227V-R229V-E252Q-I272VR117Q-M119V-A131E-L203T-I204Y- 75% G206Q-S227V-R229V-E252Q-I272VR117Q-M119V-R120V-A131E-I204Y- 83% G206Q-S227V-R229V-E252Q-I272VR117Q-M119V-R120V-A131E-L203T- 80% I204Y-S227V-R229V-E252Q-I272VR117Q-M119V-R120V-A131E-L203T- 74% I204Y-G206Q-R229V-E252Q-I272VR117Q-M119V-R120V-A131E-L203T- 85% I204Y-G206Q-S227D-R229V-E252Q-I272VR117Q-M119V-R120V-A131E-L203T- 84% I204Y-G206Q-S227V-E252Q-I272VR117Q-M119V-R120V-A131E-L203T- 73% I204Y-G206Q-S227V-R229V-I272VR117Q-M119V-R120V-A131E-L203T- 81% I204Y-G206Q-S227V-R229V-E252QR117Q-M119V-R120V-A131E-L203T- 86% I204Y-G206Q-S227V-R229V-E252Q-I272PA131E-L203K-G206Q-R229V-E252Q-I272V 61%A131E-L203T-G206Q-R229V-E252Q-I272V 52% A131E-L203K-R229V-E252Q-I272V58% A131E-L203K-G206Q-E252Q-I272V 54% L203T-G206Q-R229V-E252Q-I272V 39%A131E-L203T-G206Q-R229V-I272V 35% S095Q-A131E-L203K-G206Q-R229V- 63%K247R-E252Q-I272V S095Q-L203K-G206Q-R229V-K247R- 52% E252Q-I272VS095Q-A131E-L203T-G206Q-R229V- 60% K247R-E252Q-I272VS095Q-A131E-G206Q-R229V-K247R- 42% E252Q-I272VS095Q-A131E-L203K-R229V-K247R- 64% E252Q-I272VS095Q-A131E-L203K-G206Q-K247R- 61% E252Q-I272VS095Q-A131E-L203K-G206Q-R229V- 65% E252Q-I272VS095Q-A131E-L203K-G206Q-R229V- 49% K247R-I272VS095Q-A131E-L203K-G206Q-R229V- 62% K247R-E252QA131E-L203S-G206Q-R229V-I272P 59% L203S-G206Q-R229V-I272P 51%A131E-L203S-R229V-I272P 62% A131E-L203S-G206Q-I272P 60%A131E-L203S-G206Q-R229V 38% R117Q-L203T-A224S-E252Q 51%R117Q-A131E-L203S-I204Y-S227V- 84% R229Q-E252Q-I272PA131E-L203S-I204Y-S227V-R229Q- 73% E252Q-I272PR117Q-L203S-I204Y-S227V-R229Q- 78% E252Q-I272PR117Q-A131E-I204Y-S227V-R229Q- 82% E252Q-I272PR117Q-A131E-L203S-S227V-R229Q- 85% E252Q-I272PR117Q-A131E-L203S-I204Y-R229Q- 71% E252Q-I272PR117Q-A131E-L203S-I204Y-S227V- 84% R229Q-I272PR117Q-A131E-L203S-I204Y-S227V- 82% R229Q-E252QA131E-L203S-G206Q-A217Y-R229V- 86% I272P R117Q-M119V-R120V-A131E-L203T-91% I204Y-G206Q-A217Y-S227D-R229V- E252Q R117Q-M119V-R120V-A131E-L203T-93% I204Y-G206Q-A217Y-S227D-R229V- E252Q-I272VR117Q-M119V-R120V-A131E-L203T- 93% I204Y-G206Q-A217Y-S227V-R229V-E252Q-I272P R117Q-M119V-R120V-A131E-L203T- 88%I204Y-A205W-G206Q-S227D-R229V- E252Q R117Q-M119V-R120V-A131E-L203T- 92%I204Y-A205W-G206Q-S227D-R229V- E252Q-I272VR117Q-M119V-R120V-A131E-L203T- 93% I204Y-A205W-G206Q-S227V-R229V-E252Q-I272P R117E-Y127D-A131E-I204Y-A205W- 100%  A217Y-V221T-R229C-E252QR117G-Y127D-A131E-I204Y-A217Y- 78% V221T-R229Q-E252QR117G-Y127D-A131E-I204Y-A205W- 87% A217Y-V221T-R229Q-E252QR117G-Y127D-A131E-A217Y-V221T- 87% E252Q-I272KR117G-Y127D-A131E-A205W-A217Y- 97% V221T-E252Q-I272KR117G-A131E-I204Y-A217Y-V221T- 70% R229Q-E252QR117G-A131E-I204Y-A205W-A217Y- 86% V221T-R229Q-E252QR117G-A131E-I204Y-G209A-A217Y- 84% V221T-R229Q-E252Q-I272KR117G-A131E-I204Y-A205W-G209A- 92% A217Y-V221T-R229Q-E252Q-I272KR117E-A131E-A205W-A217Y-V221T- 95% E252Q Y127D-A131E-I204Y-A205W-G209A-87% A217Y-V221T-R229Q R117E-A131E-A217Y 92% A131E-A217Y 90% L203R-A217Y87% L203R-G209D-A217Y 91% G209D-A217Y 89% Y022F-R117Q-L203R-G209D-A217Y93% Y022F-A159D-L203R-G206C-K208Q- 89% G209D-A217Y-I272PR117E-A131E-I204Y-G209A-A217Y 62% R117E-A131E-I204Y-G209A-A217Y-V221T82% R117E-A131E-I204Y-G209A-A217Y- 92% V221T-R229C-E252Q-I272KR117E-A131E-F143L-I204Y-G209A- 88% A217Y-V221T-R229C-E252Q-I272KY022F-L203R-G206C-K208Q-G209D-A217Y 90% Y022F-R117E-A131E-L203R-G209D80% Y022F-R117E-A131E-L203R-G209D-A217Y 87% R117E-A131E-L203R-G209D 77%R117E-A131E-L203R-G209D-A217Y 92% R117E-A131E-L203R-I204Y-G209A-V221T89% R117E-A131E-L203R-I204Y-G209A- 92% A217Y-V221T A217Y-E252Q 70%R117E-A131E-I204Y-G209A-A217Y-E252Q 76% Y022F-R053Q-R117Q-F143L-L203R-85% G206C-K208Q-R229V-E252Q Y022F-R117D-F143L-I272P 71%Y022F-R117E-A131E-A217Y-V221T 94% Y022F-R117E-A131E-F143L-A217Y-V221T93% A131E-L203R-A217Y-I272P 89% A131E-F143L-L203R-A217Y-I272P 90%Y022F-R053Q-F143L-R229V-E252Q-I272P 71%R117E-A131E-A217Y-V221T-E252Q-I272K 91%R117E-Y127D-A131E-A217Y-E252Q-I272K 92%R117E-Y127D-A131E-A217Y-V221T-E252Q 95%R117E-Y127D-A131E-A217Y-V221T-I272K 84%R117E-Y127D-A131E-I204Y-G209A-A217Y- 84% V221T-E252Q-I272KR117E-Y127D-A131E-F143L-I204Y-G209A- 92% A217Y-V221T-R229C-E252Q-I272KY127D-A217Y 86% R117E-A131E-A217Y-V221T 92%Y022F-A159D-L203R-G209D-A217Y-V221T- 94% I272KY022F-P125E-A131E-I179K-L203R-G209D- 90% A217Y-V221T-R229C-I272KA131E-A217Y-V221T 87% A131E-G209D-V221T-I272K 13%A131E-L203R-G209D-A217Y-V221T 91% A131E-L203R-G209D-A217Y-V221T-R229C-90% I272K R117E-Y127D-L203S-A217Y-E252Q 98% Y022F-A217Y-E252Q 75%Y022F-L203R-G206C-G209D-A217Y-E252Q 88%R117Q-A131E-L203S-G209D-A217Y-E252Q 89% R117Q-Y127D-A217Y-E252Q 87%A131D-R229V-E252Q-I272P 51% Y022F-A131D-A217Y-R229V-E252Q-I272P 87%Y022F-A131E-R229V-E252Q-I272P 48% Y022F-A131D-R229V-E252Q-I272K 55%Y022F-A131E-R229V-E252Q-I272K 46% R117E-Y127D-A131D-A217Y-V221T- 98%E252Q-I272P R117E-Y127D-A131D-A217Y-V221T- 94% R229C-E252Q-I272PR117E-Y127D-A131D-A217Y-V221T- 99% R229V-E252Q-I272PY127D-A131E-A217Y-V221T-R229V- 81% E252Q-I272KY127D-A131E-A217Y-V221T-R229C- 75% E252Q-I272KY127D-A131E-A159D-A217Y-V221T- 85% R229C-E252Q-I272K L203R-R229V-E252Q51% L203R-G209D-A217Y-E252Q 89% L203R-G209A-A217Y-E252Q 89%L203R-A217Y-R229V 88% L203R-G209D-A217Y-R229Q-E252Q 88%L203R-G209D-A217Y-R229V-E252Q 90% A159D-L203R-G209D-A217Y-E252Q 95%A159D-L203R-G209D-A217Y 92% A159D-L203R-G209D-A217Y-I272K 91%Y022F-L203R-G209D-A217Y 90% Y022F-L203R-G209D-A217Y-E252Q 85%Y127D-A131E-I204Y-A217Y-V221T- 71% R229C-E252Q-I272KY127D-A131E-L203R-A217Y-V221T- 82% R229C-E252Q-I272KL203S-G209D-A217Y-R229A 89% L203N-A217Y-A224S-R229A 90%R117Q-L203S-A217Y-A224S- 93% S227D-R229S R117Q-K121S-L203R-R229C 51%R117Q-A131E-L203R 58% L203C-A217Y-A224S-R229P 84%R117Q-K121S-L203C-G206E-A217Y- 97% S227D-R229SA131E-L203R-G209D-A224S-R229P 71% R120Q-A131E-L203R 53%R117Q-L203R-A217Y-A224S-S227D- 94% R229A A217Y-A224S-R229A 89%L203R-S227D-R229Q 50% A131E-L203R-G209D-R229S 66% K121S-L203R 37%R117S-L203C-G206E-A217Y-R229Q 88% R117E-L203R-G209D-A224S-R229S 72%R120Q-L203C-G209D-R229S 43% L203R-K208E-G209D-A224S 40%R117S-R120Q-K121S-L203R-A217Y- 94% A224S-R229C L203R-A224S 49%R117Q-A131E-L203C-A217Y- 90% S227D-R229P R117S-L203S-A224S 38%Y127E-L203S-A217Y-R229C 89% R117Q-L203R-G206E-G209D- 92% A217Y-S227DR117S-K121S-L203R-R229P 53% R117S-R120Q-A131E 10% R117E-G209D 31%R117S-R120Q-Y127E-L203R- 91% G206E-A217Y L203R-A217Y-A224S-R229Q 93%R120Q-G206E-G209D-A217Y-A224S 94% R117E-R120Q-L203R-G209D- 77%A217Y-A224S A131E-G209D-A217Y-A224S-R229A 90% G209D-A224S 12%R117S-Y127E-A217Y-A224S- 93% S227D-R229C K121E-L203N-A217Y-R229A 92%Y127E-A217Y-A224S-R229S 90% R117S-A131E-L203S-R229S 52%L203R-K208E-R229A 19% R117Q-A131K-L203R-A224S 56% L203R-R229A 37%R120E-L203R-R229P 50% R120Q-A217Y 91% R120Q-L203N-A217Y-A224S-R229S 89%L203R-A217Y-A224S-R229A 91% L203S-K208E-G209D-A217Y- 92%A224S-S227D-R229S R117S-G209D-A217Y 89% L203S-G209D-S227D 51%A131E-L203R-A224S-R229A 63% L203R-A224S-R229P 49%Y127E-L203R-A217Y-R229C 92% R117Q-L203R-R229S 48%K121S-L203R-A217Y-A224S 91% R117S-R120Q-L203R-R229Q 57%R117S-R120Q-L203R 51% R117S-L203R-R229A 49% R117S-L203R-A224S 58%R117Q-S227D-R229A 57% L203C-G209D-A217Y-S227D 90% R120Q-L203R-A217Y 89%R117S-L203R-A224S-R229C 64% K121S-L203C-R229A 24%K121E-L203N-A217Y-A224S- 97% S227D-R229P K121E-L203R-A217Y 104% A131E-G209D-A217Y 91% L203N-A224S-S227D-R229C 20% K121E-L203R-R229C 51%R117Q-L203R-G209D-R229A 66% R117Q-A224S-R229C 32%R117S-L203R-A224S-R229A 56% R117Q-K121S-L203R-S227D-R229P 67%R117S-Y127E-L203S-A224S-S227D 95% R117S-L203R-R229Q 48% R117Q-L203R 46%A131E-L203R-A224S-R229S 60% K121S-A131E-L203R-A224S 66%R117S-K121S-L203C-G206E- 95% A217Y-A224S-S227D R117Q-G209D 19%R120Q-A131E-L203R-R229C 54% R117Q-R120Q-Y127E-L203S- 96% A217Y-A224SR117Q-R120Q-L203R-A217Y-A224S 95% R117Q-R120Q-L203R-A217Y 96%R120Q-L203R-A217Y-A224S- 92% S227D-R229S R117Q-R120E-L203R-R229Q 62%R117Q-R120E-L203R-A217Y-A224S 96% A131E-L203R-A224S 57%R117S-A131E-L203N-S227D 39% L203S-R229C 62% L203S-A224S-S227D-R229A 34%L203R-G209D-R229C 54% L203R-A217Y-R229A 88%L203R-G206E-A217Y-S227D-R229P 93% L203R-A217Y-R229S 91%R117Q-K121E-L203R-A224S-S227D 85% R117S-K121S-L203C-G206E- 97%A217Y-A224S-S227D-R229S R117S-K121S-L203N-A217Y-R229Q 95%R117Q-L203S-R229C 53% R117Q-L203R-G206E-A224S-R229S 59%R117Q-R120E-L203R 59% R120E-G206E-A217Y-R229Q 91% R117S-L203S-K208E 23%R120Q-K121E-L203C-A217Y-R229A 73% A131E-L203R-A217Y-A224S-R229A 97%R120E-L203R-G209D-R2290 65% A131E-L203R-S227D-R229P 62%L203R-G209D-A217Y-A224S-R229S 92% L203N-G206E-A217Y 87%R120Q-L203R-G206E-A217Y-R229Q 95% R117Q-K121S-L203R-G206E- 94%A217Y-S227D-R229Q R120E-L203R-S227D-R229A 62% R117S-L203R-A224S-R229S59% R120Q-L203R-A224S-R229C 59% L203R-R229S 39% L203R-R229P 40%R117Q-K121E-L203R-R229P 71% R117E-R120Q-L203R-A224S 65%R120Q-A131E-L203C-A217Y-A224S-R229S 92%R117Q-R120Q-L203S-A217Y-A224S-R229C 97% R117Q-A131E-L203R-G206E- 96%A217Y-A224S-S227D-R229A R120Q-K121E-L203C-A217Y-A224S 84%A131E-L203N-A217Y-A224S-S227D 94% R117E-L203R-G209D 66%R120Q-L203C-G206E 25% L203R-G209D-S227D 65%R117S-L203C-G206E-A217Y-A224S 91% R117E-K121E-L203R 51%R117E-A131E-L203N-G209D-A217Y 92% L203C-S227D 15% L203S-A217Y-R229Q 88%R117E-A217Y-S227D 89% R117Q-K121S-L203R-R229Q 45% L203R-K208E-G209D 51%R117S-L203S-G206E-G209D 64% R117S-K121E-L203R-A224S-R229P 70%L203C-A217Y-S227D-R229C 86% R117Q-K121S-L203R 29%R117Q-R120Q-Y127E-L203R-A217Y-A224S 81% K121E-L203S-A217Y-R229P 91%R120Q-L203R-G206E-A217Y-A224S 90% I154V-K170E-A217Y 91% V039I-A217Y 90%A217Y-V265A 10% Y026C-K208E-A217Y 52% E067G-I204V-A217Y 78% K170E-A217Y90% K134E-A217Y 84% A217Y-V235A 17% F060L-A217Y-E252G 70%I064T-A217Y-V261A 55% I111T-A217Y 62% M166T-A217Y 86% A093S-R120S-A217Y73% K170S-A217Y 94% S141N-A217Y-A218D-E252D 42% S141N-A217Y-E252G 37%I019L-N149I-F196L-A217Y 69% H011R-I064V-K134M-E163G-A217Y-E252V 10%

Example 4 Lipolytic Enzyme Variants with Improved Stability in LiquidDetergents

The stability of lipase variants of the parent lipase of SEQ ID NO:1described herein was determined as described in Example 2 for Stabilityin HDL. Lipase samples were incubated in a 50% final concentration ofheat-treated Persil Color Gel detergent for a 48-hour period of heatstress at 40° C. Lipase activity was subsequently measured as describedin Example 2. The reaction emulsion to measure activity consisted of:1.3 mM 4-methylumbelliferyl-caprylate, 10 mM HEPES pH8, 100 mM NaCl, 1mM CaCl₂), 0.2% Triton X100, and 0.2% bovine serum albumin. Activitylevels measured for unstressed controls were compared to activity levelsafter heat stress. The ratio of stressed vs unstressed values was usedto calculate the % residual activity and compared to the % residualactivity of the parent lipase (SEQ ID NO:1). The average % residualactivity for the parent lipase (SEQ ID NO:1) under these conditions was6%.

Lipase variants are referred to by their positional modificationrelative to the lipase of SEQ ID NO:1. For example, lipase variant“N146H_N280K” is a variant of SEQ ID NO: 1 that comprises a substitutionat position 146 (N changed to H in variant) and a substitution atposition 280 (N changed to K in variant). Lipase variants with animproved detergent stability compared to the parent lipase of SEQ ID NO:1 are shown in Table 21.

TABLE 21 Lipase variants with Improved Detergent Stability inHeat-Treated Commercial Liquid Detergent. % Lipase variant (positionsmodified relative Residual to parent of SEQ ID NO: 1) Activity SEQ IDNO: 1  6% T004K-A070V-K071E-R117Q-A131T- 58%F143L-N146H-A159D-I179K-L203R- G206E-G209D-L212R-R229H-N280K Y022F-L203R82% L203R-I204Y 41% L203R-K208Q 23% L203R-G209D 32% L203R-R229Q 42%L203R-I272P 30% YO22F-R117D-L203R 18% YO22F-R117Q-L203R 61%YO22F-A131E-L203R 48% Y022F-L203R-G206C 47% Y022F-L203R-K208Q 64%Y022F-L203R-G209D 57% Y022F-L203R-G209A 81% Y022F-L203R-R229Q 54%Y022F-L203R-R229T 39% Y022F-L203R-I272P 58% A131D-L203R-E252Q 65%R117Q-L203R-K208Q 39% A131E-L203R-K208Q 22% L203R-I204Y-K208Q 27%L203R-K208Q-G209D 55% L203R-K208Q-G209A 61% L203R-K208Q-E252Q 69%L203R-K208Q-V261L 37% L203R-K208Q-A262M 23% L203R-K208Q-I272P 27%A131E-L203R-G209D 27% L203R-I204Y-G209D 71% L203R-G206C-G209D 49%L203R-G209D-R229Q 30% L203R-G209D-E252Q 75% L203R-G209D-I272P 50%R117Q-L203R-R229T 75% L203R-I204Y-R229T 63% L203R-G209A-R229T 80%L203R-R229T-E252Q 51% L203R-R229T-A262M 42% L203R-R229T-I272P 33%R117Q-L203R-I272P 34% Y127D-L203R-I272P 62% A131E-L203R-I272P 35%L203R-I204Y-I272P 59% L203R-G206C-I272P 22% L203R-G209A-I272P 40%L203R-E252Q-I272P 88% A131D-L203R-G209D-R229T 68%A131D-L203S-R229T-I272P 58% L203R-K208Q-G209D-R229T 39%L203R-K208Q-R229T-I272P 48% L203S-K208Q-G209D-R229T 51%L203S-K208Q-R229T-I272P 40% Y022F-R117Q-A131T-F143L-L203R- 67%G209D-R229Q Y199H-L203R-G209D-A217Y-R229Q 97% L203R-G209D-A217Y-R229Q84% R117Q-L203R-G209D-R229C 54% A217Y-V221T-R229C-E252Q-A262M- 61% I272PY022F-A159D-L203R-G206C-K208Q- 78% G209D-I272PY022F-L203R-G206C-K208Q-G209D 52% P125E-L203R-K208Q-G209D 37%T004K-K071E-R117D-A131T-F143L- 58% N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229C-N280K T004K-K071E-R117Q-A131D-F143L- 65%N146H-A159D-I179K-L203R-G206E- G209D-L212R-R229C-N280KA159D-L203R-G206E-G209D 48% YO22F-R117Q-A131D-L203R-R229V- 54% E252QY022F-R053Q-R117Q-E252Q-V261L- 52% I272P Y022F-R117Q-A131D-E252Q 51%Y022F-R117Q-R229V-E252Q-V261L- 70% I272P Y022F-R053Q-R229V-E252Q-I272P33% R117Q-A131D-E252Q-I272P 67% Y022F-R053Q-R117Q-R229V-V261L 58%Y022F-R117Q-G209D-R229V-E252Q- 78% I272P Y022F-A131D-R229V-E252Q-I272P108%  Y022F-R053Q-R117Q-L203R-R229V- 88% E252Q-V261LY022F-R053Q-R117Q-L203R-R229V- 97% E252Q-I272PY022F-R053Q-R117Q-L203R-R229V- 88% E252Q Y022F-A131D-L203R-R229V-E252Q-90% I272P Y022F-R053Q-R117Q-E252Q-I272P 70% Y022F-R117Q-E252Q-V261L 69%Y022F-R117Q-R229V-I272P 78% Y022F-R117D-I272P 82%R117E-A131E-I204Y-E252Q-I272K 82% R117E-A131E-I204Y-G209A-V221T- 59%R229C R117E-A131E-R229C 75% Y127D-G209A-V221T-R229C 51%R117E-Y127D-A131E-I204Y-A217Y- 44% V221T-R229C-E252QY127D-A131E-A217Y-V221T-E252Q 93% Y127D-A131E-I204Y-G209A-V221T- 90%I272K Y127D-V221T-R229C-E252Q-I272K 94% R117E-Y127D-A131E-A217Y-V221T-79% E252Q-I272K G209A-V221T-R229C-E252Q 64%Y127D-I204Y-V221T-R229C-I272K 61% Y127D-A131E-I204Y-V221T-R229C- 98%E252Q-I272K R117E-A131E-I204Y-G209A-V221T- 74% E252Q-I272KA131E-G209A-V221T-R229C 31% R117E-A131E-I204Y-G209A-V221T- 65% I272KR117E-A131E-I204Y-E252Q 124%  R117E-A131E-I204Y-V221T-R229C- 82% E252QR117E-A131E-I204Y-G209A-V221T- 82% R229C-E252Q-I272KR117E-V221T-R229C-E252Q 55% Y127D-A131E-I204Y-G209A-R229C- 87%E252Q-I272K R117E-A131E-I204Y-I272K 23% R117E-A131E-I204Y-V221T-R229C-64% I272K R117E-A131E-A217Y-V221T-E252Q 59% R117E-A131E-R229C-E252Q 71%Y127D-A131E-I204Y-G209A-V221T- 55% R229C Y127D-A131E-G209A-R229C-E252Q-61% I272K R117E-I204Y-G209A-V221T-E252Q 76%R117E-A131E-G209A-V221T-R229C- 93% E252Q Y127D-I204Y-G209A-E252Q-I272K45% Y127D-I204Y-V221T-R229C 77% R117E-A131E-V221T-I272K 72%M119V-L203S-E252Q 60% M119V-L203S-S227V-E252Q-I272P 69%R117Q-M119V-L203S-S227V-E252Q- 70% I272P M119V-R120V-A131E-L203T-I204Y-63% G206Q-S227V-R229V-E252Q-I272V R117Q-R120V-A131E-L203T-I204Y- 67%G206Q-S227V-R229V-E252Q-I272V R117Q-M119V-A131E-L203T-I204Y- 66%G206Q-S227V-R229V-E252Q-I272V R117Q-M119V-R120V-A131E-I204Y- 74%G206Q-S227V-R229V-E252Q-I272V R117Q-M119V-R120V-A131E-L203T- 69%I204Y-S227V-R229V-E252Q-I272V R117Q-M119V-R120V-A131E-L203T- 67%I204Y-G206Q-R229V-E252Q-I272V R117Q-M119V-R120V-A131E-L203T- 69%I204Y-G206Q-S227D-R229V-E252Q- I272V R117Q-M119V-R120V-A131E-L203T- 71%I204Y-G206Q-S227V-E252Q-I272V R117Q-M119V-R120V-A131E-L203T- 22%I204Y-G206Q-S227V-R229V-I272V R117Q-M119V-R120V-A131E-L203T- 68%I204Y-G206Q-S227V-R229V-E252Q R117Q-M119V-R120V-A131E-L203T- 76%I204Y-G206Q-S227V-R229V-E252Q- I272P R117Q-A131E-L203S-I204Y-S227V- 78%R229Q-E252Q-I272P A131E-L203S-I204Y-S227V-R229Q- 71% E252Q-I272PR117Q-L203S-I204Y-S227V-R229Q- 71% E252Q-I272PR117Q-A131E-I204Y-S227V-R229Q- 69% E252Q-I272PR117Q-A131E-L203S-S227V-R229Q- 72% E252Q-I272PR117Q-A131E-L203S-I204Y-R229Q- 76% E252Q-I272PR117Q-A131E-L203S-I204Y-S227V- 51% R229Q-I272PR117Q-A131E-L203S-I204Y-S227V- 65% R229Q-E252QA131E-L203S-G206Q-A217Y-R229V- 81% I272P R117Q-M119V-R120V-A131E-L203T-82% I204Y-G206Q-A217Y-S227D-R229V- E252Q R117Q-M119V-R120V-A131E-L203T-79% I204Y-G206Q-A217Y-S227D-R229V- E252Q-I272VR117Q-M119V-R120V-A131E-L203T- 84% I204Y-G206Q-A217Y-S227V-R229V-E252Q-I272P R117Q-M119V-R120V-A131E-L203T- 85%I204Y-A205W-G206Q-S227D-R229V- E252Q R117Q-M119V-R120V-A131E-L203T- 83%I204Y-A205W-G206Q-S227D-R229V- E252Q-I272VR117Q-M119V-R120V-A131E-L203T- 83% I204Y-A205W-G206Q-S227V-R229V-E252Q-I272P R117E-Y127D-A131E-I204Y-A205W- 65% A217Y-V221T-R229C-E252QR117G-Y127D-A131E-I204Y-A217Y- 75% V221T-R229Q-E252QR117G-Y127D-A131E-I204Y-A205W- 74% A217Y-V221T-R229Q-E252QR117G-Y127D-A131E-A217Y-V221T- 82% E252Q-I272KR117G-Y127D-A131E-A205W-A217Y- 80% V221T-E252Q-I272KR117G-A131E-I204Y-A205W-A217Y- 79% V221T-R229Q-E252QR117G-A131E-I204Y-G209A-A217Y- 76% V221T-R229Q-E252Q-I272KR117G-A131E-I204Y-A205W-G209A- 87% A217Y-V221T-R229Q-E252Q-I272KR117E-A131E-A205W-A217Y-V221T- 76% E252Q Y127D-A131E-I204Y-A205W-G209A-65% A217Y-V221T-R229Q R117E-A131E-A217Y 79% A131E-A217Y 72% L203R-A217Y75% L203R-G209D-A217Y 73% G209D-A217Y 71% Y022F-R117Q-L203R-G209D-A217Y79% R117E-A131E-I204Y-G209A-A217Y- 58% V221TR117E-A131E-I204Y-G209A-A217Y- 80% V221T-R229C-E252Q-I272KR117E-A131E-F143L-I204Y-G209A- 76% A217Y-V221T-R229C-E252Q-I272KY022F-L203R-G206C-K208Q-G209D- 61% A217Y Y022F-R117E-A131E-L203R-G209D-80% A217Y R117E-A131E-L203R-G209D 37% R117E-A131E-L203R-G209D-A217Y 79%R117E-A131E-L203R-I204Y-G209A- 71% V221T R117E-A131E-L203R-I204Y-G209A-84% A217Y-V221T R117E-A131E-I204Y-G209A-A217Y- 64% E252QY022F-R053Q-R117Q-F143L-L203R- 76% G206C-K208Q-R229V-E252QY022F-R117D-F143L-I272P 41% Y022F-R117E-A131E-A217Y-V221T 80%Y022F-R117E-A131E-F143L-A217Y- 79% V221T A131E-L203R-A217Y-I272P 78%A131E-F143L-L203R-A217Y-I272P 80% Y022F-R053Q-F143L-R229V-E252Q- 68%I272P R117E-A131E-A217Y-V221T-E252Q- 73% I272KR117E-Y127D-A131E-A217Y-E252Q- 78% I272K R117E-Y127D-A131E-A217Y-V221T-69% E252Q R117E-Y127D-A131E-F143L-I204Y- 80%G209A-A217Y-V221T-R229C-E252Q- I272K Y127D-A217Y 74%R117E-A131E-A217Y-V221T 77% Y022F-A159D-L203R-G209D-A217Y- 90%V221T-I272K Y022F-P125E-A131E-I179K-L203R- 85%G209D-A217Y-V221T-R229C-I272K A131E-A217Y-V221T 77%A131E-L203R-G209D-A217Y-V221T 84% A131E-L203R-G209D-A217Y-V221T- 79%R229C-I272K R117E-Y127D-L203S-A217Y-E252Q 70% Y022F-A217Y-E252Q 59%Y022F-L203R-G206C-G209D-A217Y- 69% E252Q R117Q-A131E-L203S-G209D-A217Y-73% E252Q R117Q-Y127D-A217Y-E252Q 86% R117E-Y127D-A131D-A217Y-V221T- 91%E252Q-I272P Y127D-A131E-A217Y-V221T-R229V- 77% E252Q-I272KY127D-A131E-A217Y-V221T-R229C- 75% E252Q-I272KY127D-A131E-A159D-A217Y-V221T- 67% R229C-E252Q-I272KL203R-G209D-A217Y-E252Q 79% L203R-G209A-A217Y-E252Q 74%L203R-A217Y-R229V 70% L203R-G209D-A217Y-R229Q-E252Q 73%L203R-G209D-A217Y-R229V-E252Q 71% A159D-L203R-G209D-A217Y-E252Q 67%A159D-L203R-G209D-A217Y 75% A159D-L203R-G209D-A217Y-I272K 63%Y022F-L203R-G209D-A217Y 75% Y022F-L203R-G209D-A217Y-E252Q 68%Y127D-A131E-I204Y-A217Y-V221T- 73% R229C-E252Q-I272KY127D-A131E-L203R-A217Y-V221T- 74% R229C-E252Q-I272KL203S-G209D-A217Y-R229A 72% L203N-A217Y-A224S-R229A 72%R117Q-L203S-A217Y-A224S-S227D- 75% R229S L203C-A217Y-A224S-R229P 59%R117Q-K121S-L203C-G206E-A217Y- 66% S227D-R229SR117Q-L203R-A217Y-A224S-S227D-R229A 69% A217Y-A224S-R229A 67%R117S-L203C-G206E-A217Y-R229Q 71% R117E-L203R-G209D-A224S-R229S 25%R117Q-A131E-L203C-A217Y-S227D-R229P 75% Y127E-L203S-A217Y-R229C 71%R117Q-L203R-G206E-G209D-A217Y-S227D 75%R117S-R120Q-Y127E-L203R-G206E-A217Y 71% L203R-A217Y-A224S-R229Q 69%R120Q-G206E-G209D-A217Y-A224S 75% A131E-G209D-A217Y-A224S-R229A 74%R117S-Y127E-A217Y-A224S-S227D-R229C 69% K121E-L203N-A217Y-R229A 65%Y127E-A217Y-A224S-R229S 70% R120Q-A217Y 69%R120Q-L203N-A217Y-A224S-R229S 72% L203R-A217Y-A224S-R229A 69%L203S-K208E-G209D-A217Y-A224S- 71% S227D-R229S R117S-G209D-A217Y 73%Y127E-L203R-A217Y-R229C 69% K121S-L203R-A217Y-A224S 57%L203C-G209D-A217Y-S227D 65% R120Q-L203R-A217Y 57%K121E-L203N-A217Y-A224S-S227D-R229P 69% K121E-L203R-A217Y 63%A131E-G209D-A217Y 73% R117S-Y127E-L203S-A224S-S227D 69%R117S-K121S-L203C-G206E-A217Y- 70% A224S-S227DR117Q-R120Q-Y127E-L203S-A217Y-A224S 67% R117Q-R120Q-L203R-A217Y-A224S61% R117Q-R120Q-L203R-A217Y 63% R120Q-L203R-A217Y-A224S-S227D-R229S 69%R117Q-R120E-L203R-A217Y-A224S 59% L203R-A217Y-R229A 69%L203R-G206E-A217Y-S227D-R229P 70% L203R-A217Y-R229S 72%R117S-K121S-L203C-G206E-A217Y- 57% A224S-S227D-R229SR117S-K121S-L203N-A217Y-R229Q 76% R120E-G206E-A217Y-R229Q 78%R120Q-K121E-L203C-A217Y-R229A 107%  A131E-L203R-A217Y-A224S-R229A 94%R120E-L203R-G209D-R229Q 50% A131E-L203R-S227D-R229P 10%L203R-G209D-A217Y-A224S-R229S 64% L203N-G206E-A217Y 64%R120Q-L203R-G206E-A217Y-R229Q 68% R117Q-K121S-L203R-G206E-A217Y- 81%S227D-R229Q R120E-L203R-S227D-R229A 35% R117S-L203R-A224S-R229S 14%R120Q-L203R-A224S-R229C 13% R117Q-K121E-L203R-R229P 49%R117E-R120Q-L203R-A224S 37% R120Q-A131E-L203C-A217Y-A224S-R229S 76%R117Q-R120Q-L203S-A217Y-A224S-R229C 80% R117Q-A131E-L203R-G206E-A217Y-85% A224S-S227D-R229A R120Q-K121E-L203C-A217Y-A224S 105% A131E-L203N-A217Y-A224S-S227D 95% R117E-L203R-G209D 54%L203R-G209D-S227D 27% R117S-L203C-G206E-A217Y-A224S 66%R117E-A131E-L203N-G209D-A217Y 80% L203S-A217Y-R229Q 80%R117E-A217Y-S227D 74% R117S-L203S-G206E-G209D 35%R117S-K121E-L203R-A224S-R229P 62% L203C-A217Y-S227D-R229C 71%R117Q-R120Q-Y127E-L203R-A217Y-A224S 72% K121E-L203S-A217Y-R229P 66%R120Q-L203R-G206E-A217Y-A224S 78% I154V-K170E-A217Y 64% K170E-A217Y 47%K134E-A217Y 63% M166T-A217Y 65% K170S-A217Y 59% I019L-N149I-F196L-A217Y57%

Example 5 Wash Performance of Lipase Variants

In order to assess the cleaning performance of lipase variants of theparent lipase of SEQ ID NO:1 disclosed herein, the wash performance wasdetermined using a miniswatch assay as described herein, where a washperformance, PI(wash) was determined. The wash performance of the lipasevariants at 0.3 ppm was determined based on the % OSRI (cleaning)against CS-61 (Beef fat on cotton) using the miniswatch assay describedherein (Example 2) at 16° C. in Persil Color Gel using CS-61, 250 ppm3:1 Ca²⁺:Mg²⁺ hardness, 30 min, 10 mM HTEPES, pH 8.0. The washperformance index (PI(wash)) of a lipase variant relative to a referencelipase (SEQ ID NO: 1) was calculated as:

PI(Wash)=((% SRI(lipase variant)−% OSRI(no enzyme))/((% SRI(lipasereference)−% OSRI (no enzyme)).

Lipase variants are referred to by their positional modificationrelative to the lipase of SEQ ID NO:1. For example, lipase variant“N146H_N280K” is a variant of SEQ ID NO: 1 that comprises a substitutionat position 146 (N changed to H in variant) and a substitution atposition 280 (N changed to K in variant). Wash performance of lipasevariants is shown in Table 22.

TABLE 22 Wash Performance Index of lipase variants of SEQ ID NO: 1 at0.3 ppm Wash Lipase variant (based on positions performance modifiedrelative to parent of index, PI SEQ ID NO: 1) (wash) SEQ ID NO: 1 1.00T004K-A070V-K071E-R117Q-A131T- 1.43 F143L-N146H-A159D-I79K-L203R-G206E-G209D-L212R-R229H-N280K Y022F-L203R 0.56 L203R-I204Y 1.04L203R-K208Q 1.34 L203R-G209D 1.23 L203R-R229Q 1.09 L203R-I272P 1.23Y022F-R117D-L203R 0.93 Y022F-R117Q-L203R 0.09 Y022F-A131E-L203R 0.95Y022F-L203R-G206C 0.83 Y022F-L203R-K208Q 0.87 Y022F-L203R-G209D 0.76Y022F-L203R-G209A 0.00 Y022F-L203R-R229Q 1.12 Y022F-L203R-R229T 1.04Y022F-L203R-I272P 1.01 A131D-L203R-E252Q 1.28 R117Q-L203R-K208Q 1.56A131E-L203R-K208Q 1.57 L203R-I204Y-K208Q 0.78 L203R-K208Q-G209D 0.72L203R-K208Q-G209A 0.95 L203R-K208Q-E252Q 1.34 L203R-K208Q-V261L 0.81L203R-K208Q-A262M 1.20 L203R-K208Q-I272P 0.83 A131E-L203R-G209D 1.44L203R-I204Y-G209D 0.28 L203R-G206C-G209D 1.08 L203R-G209D-R2290 1.36L203R-G209D-E252Q 1.11 L203R-G209D-I272P 1.39 R117Q-L203R-R229T 1.31L203R-I204Y-R229T 0.89 L203R-G209A-R229T 0.94 L203R-R229T-E252Q 1.40L203R-R229T-A262M 1.56 L203R-R229T-I272P 1.17 R117Q-L203R-I272P 1.09Y127D-L203R-I272P 1.72 A131E-L203R-I272P 0.99 L203R-I204Y-I272P 1.78L203R-G206C-I272P 2.33 L203R-G209A-I272P 1.91 L203R-E252Q-I272P 2.07A131D-L203R-G209D-R229T 0.81 A131D-L203S-R229T-I272P 1.90L203R-K208Q-G209D-R229T 1.09 L203R-K208Q-R229T-I272P 1.28L203S-K208Q-G209D-R229T 1.96 L203S-K208Q-R229T-I272P 1.74Y022F-R117Q-A131T-F143L-L203R-G209D- 2.05 R229QY199H-L203R-G209D-A217Y-R229Q 0.73 L203R-G209D-A217Y-R2290 0.84R117Q-L203R-G209D-R229C 1.92 A217Y-V221T-R229C-E252Q-A262M-I272P 0.81Y022F-A159D-L203R-G206C-K208Q-G209D- 0.27 I272PY022F-L203R-G206C-K208Q-G209D 0.66 P125E-L203R-K208Q-G209D 2.15T004K-K071E-R117D-A131T-F143L-N146H- 0.71A159D-I179K-L203R-G206E-G209D-L212R- R229C-N280KT004K-K071E-R117Q-A131D-F143L-N146H- 0.98A159D-I179K-L203R-G206E-G209D-L212R- R229C-N280K A159D-L203R-G206E-G209D0.49 Y022F-R117Q-A131D-L203R-R229V-E252Q 0.73Y022F-R53Q-R117Q-E252Q-V261L-I272P 0.25 Y022F-R117Q-A131D-E252Q 0.57Y022F-R117Q-R229V-E252Q-V261L-I272P 0.35 Y022F-R053Q-R229V-E252Q-I272P0.66 R117Q-A131D-E252Q-I272P 1.28 Y022F-R53Q-R117Q-R229V-V261L 0.29Y022F-R117Q-G209D-R229V-E252Q-I272P 1.03 Y022F-A131D-R229V-E252Q-I272P0.74 Y022F-R053Q-R117Q-L203R-R229V-E252Q-V261L 0.08Y022F-R053Q-R117Q-L203R-R229V-E252Q-I272P 0.44Y022F-R053Q-R117Q-L203R-R229V-E252Q 1.26Y022F-A131D-L203R-R229V-E252Q-I272P 0.64 Y022F-R053Q-R117Q-E252Q-I272P1.13 Y022F-R117Q-E252Q-V261L 1.12 Y022F-R117Q-R229V-I272P 1.31Y022F-R117D-I272P 0.10 R117E-A131E-I204Y-E252Q-I272K 1.86R117E-A131E-I204Y-G209A-V221T-R229C 1.64 R117E-A131E-R229C 1.26Y127D-G209A-V221T-R229C 1.11 R117E-Y127D-A131E-I204Y-A217Y-V221T- 0.98R229C-E252Q Y127D-A131E-A217Y-V221T-E252Q 1.86Y127D-A131E-I204Y-G209A-V221T-I272K 1.77 Y127D-V221T-R229C-E252Q-I272K1.75 R117E-Y127D-A131E-A217Y-V221T-E252Q-I272K 0.98G209A-V221T-R229C-E252Q 1.51 Y127D-I204Y-V221T-R229C-I272K 1.78Y127D-A131E-I204Y-V221T-R229C-E252Q-I272K 1.40R117E-A131E-I204Y-G209A-V221T-E252Q-I272K 1.76 A131E-G209A-V221T-R229C1.52 R117E-A131E-I204Y-G209A-V221T-I272K 1.66 R117E-A131E-I204Y-E252Q1.08 R117E-A131E-I204Y-V221T-R229C-E252Q 1.46R117E-A131E-I204Y-G209A-V221T-R229C- 1.28 E252Q-I272KR117E-V221T-R229C-E252Q 1.54 Y127D-A131E-I204Y-G209A-R229C-E252Q-I272K1.42 R117E-A131E-I204Y-I272K 1.60 R117E-A131E-I204Y-V221T-R229C-I272K1.30 R117E-A131E-A217Y-V221T-E252Q 0.65 R117E-A131E-R229C-E252Q 1.20Y127D-A131E-I204Y-G209A-V221T-R229C 1.07Y127D-A131E-G209A-R229C-E252Q-I272K 1.04 R117E-I204Y-G209A-V221T-E252Q1.46 R117E-A131E-G209A-V221T-R229C-E252Q 1.50Y127D-I204Y-G209A-E252Q-I272K 1.24 Y127D-I204Y-V221T-R229C 1.70R117E-A131E-V221T-I272K 0.24 R117Q-M119V-R120V-A131E-L203T-I204Y- 1.27G206Q-S227V-E252Q-I272V R117Q-M119V-R120V-A131E-L203T-I204Y- 1.26G206Q-S227V-R229V-E252Q-I272P R117Q-A131E-L203S-I204Y-S227V-R229Q- 1.16E252Q-I272P A131E-L203S-G206Q-A217Y-R229V-I272P 0.91R117Q-M119V-R120V-A131E-L203T-I204Y- 1.18G206Q-A217Y-S227D-R229V-E252Q-I272V R117Q-M119V-R120V-A131E-L203T-I204Y-1.04 G206Q-A217Y-S227V-R229V-E252Q-I272PR117Q-M119V-R120V-A131E-L203T-I204Y- 0.88 A205W-G206Q-S227D-R229V-E252QR117Q-M119V-R120V-A131E-L203T-I204Y- 1.03A205W-G206Q-S227V-R229V-E252Q-I272P R117G-Y127D-A131E-A205W-A217Y-V221T-0.92 E252Q-I272K R117G-A131E-I204Y-A205W-G209A-A217Y- 1.17V221T-R229Q-E252Q-I272K A131E-A217Y 0.97Y022F-R117E-A131E-L203R-G209D-A217Y 0.08 R117E-A131E-L203R-G209D-A217Y0.48 R117E-A131E-L203R-I204Y-G209A-A217Y-V221T 0.97Y022F-R117E-A131E-F143L-A217Y-V221T 0.72Y022F-A159D-L203R-G209D-A217Y-V221T-I272K 0.40Y022F-P125E-A131E-I179K-L203R-G209D- 0.68 A217Y-V221T-R229C-I272KA131E-A217Y-V221T 0.89 A131E-L203R-G209D-A217Y-V221T 0.76A131E-L203R-G209D-A217Y-V221T-R229C-I272K 0.95 R117Q-Y127D-A217Y-E252Q0.97 R117E-Y127D-A131D-A217Y-V221T-E252Q-I272P 0.65L203N-A217Y-A224S-R229A 1.07 R117Q-L203S-A217Y-A224S-S227D-R229S 1.01R117S-L203C-G206E-A217Y-R229Q 1.03 R117Q-A131E-L203C-A217Y-S227D-R229P0.93 R117Q-L203R-G206E-G209D-A217Y-S227D 0.55R120Q-G206E-G209D-A217Y-A224S 0.35 A131E-G209D-A217Y-A224S-R229A 0.89K121E-L203N-A217Y-A224S-S227D-R229P 0.94 R120E-G206E-A217Y-R229Q 0.83R120Q-K121E-L203C-A217Y-R229A 0.73 A131E-L203R-A217Y-A224S-R229A 0.74R120E-L203R-G209D-R229Q 0.57 A131E-L203R-S227D-R229P 0.98L203R-G209D-A217Y-A224S-R229S 0.72 L203N-G206E-A217Y 0.92R120Q-L203R-G206E-A217Y-R229Q 0.55 R117Q-K121S-L203R-G206E-A217Y-S227D-0.50 R229Q R120E-L203R-S227D-R229A 0.80 R117S-L203R-A224S-R229S 1.09R120Q-L203R-A224S-R229C 0.76 R117Q-K121E-L203R-R229P 0.95R117E-R120Q-L203R-A224S 0.92 R120Q-A131E-L203C-A217Y-A224S-R229S 0.68R117Q-R120Q-L203S-A217Y-A224S-R229C 0.75R117Q-A131E-L203R-G206E-A217Y-A224S- 0.60 S227D-R229AR120Q-K121E-L203C-A217Y-A224S 0.64 A131E-L203N-A217Y-A224S-S227D 0.86R117E-L203R-G209D 0.63 L203R-G209D-S227D 0.91R117S-L203C-G206E-A217Y-A224S 0.69 R117E-A131E-L203N-G209D-A217Y 0.56L203S-A217Y-R229Q 0.79 R117E-A217Y-S227D 0.71 R117S-L203S-G206E-G209D0.73 R117S-K121E-L203R-A224S-R229P 0.97 L203C-A217Y-S227D-R229C 0.46R117Q-R120Q-Y127E-L203R-A217Y-A224S 0.46 K121E-L203S-A217Y-R229P 0.92R120Q-L203R-G206E-A217Y-A224S 0.38 I019L-N149I-F196L-A217Y 0.00 Nolipase 0.00

Lipase variants with improved wash performance compared to the parentlipase set forth in SEQ ID NO:1 (PI wash performance>1.0) are shown inTable 23.

TABLE 23 Lipase variants with improved wash performance compared to theparent lipase set forth in SEQ ID NO: 1 (PI wash performance >1.0) WashLipase variant (based on performance positions modified relative index,to parent of SEQ ID NO: 1) PI (wash) L203R-G206C-I272P 2.33P125E-L203R-K208Q-G209D 2.15 L203R-E252Q-I272P 2.07Y022F-R117Q-A131T-F143L-L203R-G209D- 2.05 R229Q L203S-K208Q-G209D-R229T1.96 R117Q-L203R-G209D-R229C 1.92 L203R-G209A-I272P 1.91A131D-L203S-R229T-I272P 1.9 R117E-A131E-I204Y-E252Q-I272K 1.86Y127D-A131E-A217Y-V221T-E252Q 1.86 L203R-I204Y-I272P 1.78Y127D-I204Y-V221T-R229C-I272K 1.78 Y127D-A131E-I204Y-G209A-V221T-I272K1.77 R117E-A131E-I204Y-G209A-V221T-E252Q-I272K 1.76Y127D-V221T-R229C-E252Q-I272K 1.75 L203S-K208Q-R229T-I272P 1.74Y127D-L203R-I272P 1.72 Y127D-I204Y-V221T-R229C 1.7R117E-A131E-I204Y-G209A-V221T-I272K 1.66R117E-A131E-I204Y-G209A-V221T-R229C 1.64 R117E-A131E-I204Y-I272K 1.6A131E-L203R-K208Q 1.57 R117Q-L203R-K208Q 1.56 L203R-R229T-A262M 1.56R117E-V221T-R229C-E252Q 1.54 A131E-G209A-V221T-R229C 1.52G209A-V221T-R229C-E252Q 1.51 R117E-A131E-G209A-V221T-R229C-E252Q 1.5R117E-A131E-I204Y-V221T-R229C-E252Q 1.46 R117E-I204Y-G209A-V221T-E252Q1.46 A131E-L203R-G209D 1.44 T004K-A070V-K071E-R117Q-A131T-F143L- 1.43N146H-A159D-I179K-L203R- G206E-G209D-L212R-R229H-N280KY127D-A131E-I204Y-G209A-R229C-E252Q-I272K 1.42 L203R-R229T-E252Q 1.4Y127D-A131E-I204Y-V221T-R229C-E252Q-I272K 1.4 L203R-G209D-I272P 1.39L203R-G209D-R229Q 1.36 L203R-K208Q 1.34 L203R-K208Q-E252Q 1.34R117Q-L203R-R229T 1.31 Y022F-R117Q-R229V-I272P 1.31R117E-A131E-I204Y-V221T-R229C-I272K 1.3 A131D-L203R-E252Q 1.28L203R-K208Q-R229T-I272P 1.28 R117Q-A131D-E252Q-I272P 1.28R117E-A131E-I204Y-G209A-V221T-R229C- 1.28 E252Q-I272KR117Q-M119V-R120V-A131E-L203T-I204Y- 1.27 G206Q-S227V-E252Q-I272VY022F-R053Q-R117Q-L203R-R229V-E252Q 1.26 R117E-A131E-R229C 1.26R117Q-M119V-R120V-A131E-L203T-I204Y- 1.26 G206Q-S227V-R229V-E252Q-I272PY127D-I204Y-G209A-E252Q-I272K 1.24 L203R-G209D 1.23 L203R-I272P 1.23L203R-K208Q-A262M 1.2 R117E-A131E-R229C-E252Q 1.2R117Q-M119V-R120V-A131E-L203T-I204Y- 1.18G206Q-A217Y-S227D-R229V-E252Q-I272V L203R-R229T-I272P 1.17R117G-A131E-I204Y-A205W-G209A-A217Y- 1.17 V221T-R229Q-E252Q-I272KR117Q-A131E-L203S-I204Y-S227V-R229Q- 1.16 E252Q-I272PY022F-R053Q-R117Q-E252Q-I272P 1.13 Y022F-L203R-R229Q 1.12Y022F-R117Q-E252Q-V261L 1.12 L203R-G209D-E252Q 1.11Y127D-G209A-V221T-R229C 1.11 L203R-R229Q 1.09 R117Q-L203R-I272P 1.09L203R-K208Q-G209D-R229T 1.09 R117S-L203R-A224S-R229S 1.09L203R-G206C-G209D 1.08 R117E-A131E-I204Y-E252Q 1.08Y127D-A131E-I204Y-G209A-V221T-R229C 1.07 L203N-A217Y-A224S-R229A 1.07L203R-I204Y 1.04 Y022F-L203R-R229T 1.04Y127D-A131E-G209A-R229C-E252Q-I272K 1.04R117Q-M119V-R120V-A131E-L203T-I204Y- 1.04G206Q-A217Y-S227V-R229V-E252Q-I272P Y022F-R117Q-G209D-R229V-E252Q-I272P1.03 R117Q-M119V-R120V-A131E-L203T-I204Y- 1.03A205W-G206Q-S227V-R229V-E252Q-I272P R117S-L203C-G206E-A217Y-R229Q 1.03Y022F-L203R-I272P 1.01 R117Q-L203S-A217Y-A224S-S227D-R229S 1.01 SEQ IDNO: 1 1

Example 6 Detergent Stability of Lipase Variants

Crude Bacillus subtilis supernatant containing lipase wasultra-concentrated using 3 kDa molecular weight cut off filtration (Cat.No. UFC9003 EMD Millipore). The concentrated material was formulated in10 mM HEPES (pH 8.0), 250 ppm 3:1 Ca²⁺:Mg²⁺ hardness, and 16% glycerol.A 1% (v/v) of the formulated material was added to full strength modelHDL detergent, as described in Example 2, and incubated at 30° C. Thelipase activity was assessed at timepoints over a period of 7 days using4-methylumbelliferone caprylate as a substrate. The lipase samples wereeach diluted with an emulsion to a final concentration of 1.3 mM4-methylumbelliferone caprylate, 0.2% Triton X-100, 10 mM HEPES (pH8.0), 250 ppm 3:1 Ca²⁺:Mg²⁺ hardness. Samples were added to a Corning®96 Well Solid Polystyrene Microplate (Sigma Cat. No. CLS3915). Therelease of 4-methylumbelliferone due to hydrolysis was monitored bymeasuring the fluorescence with the excitation at 380 nm and theemission at 450 nm for 5 minutes in 15 second intervals with aSpectraMax (Molecular Devices). Percent lipase activity remaining in theHDL Stability study after certain times point are shown in Tables 24 and25.

TABLE 24 Lipase variants with Improved Detergent Stability in ModelLiquid Detergent. % Residual Activity Lipase variant (positions modifiedrelative Day Day Day to parent of SEQ ID NO: 1) 0 3 7 Y022F-L203R 100% 2%  1% Y022F-L203R-G209A 100%  2%  2% L203R-E252Q-I272P 100%  5%  0%Y199H-L203R-G209D-A217Y-R229Q 100% 50% 40%Y022F-A159D-L203R-G206C-K208Q-G209D-I272P 100% 56% 32%Y022F-R117Q-R229V-E252Q-V261L-I272P 100% 16%  2%Y022F-A131D-R229V-E252Q-I272P 100% 13%  2%Y022F-R053Q-R117Q-L203R-R229V-E252Q-I272P 100% 56% 38%Y127D-A131E-A217Y-V221T-E252Q 100% 61% 44% Y127D-V221T-R229C-E252Q-I272K100%  1%  0% R117E-A131E-G209A-V221T-R229C-E252Q 100% 10%  3%L203R-G209D-A217Y-R229Q 100% 79% 80%R117E-Y127D-A131E-A217Y-V221T-E252Q-I272K 100% 72% 67% SEQID NO: 1 100% 0%  0%

TABLE 25 Lipase variants with Improved Detergent Stability in ModelLiquid Detergent. % Residual Activity Lipase variant (positions modifiedrelative Day Day Day to parent of SEQ ID NO: 1) 0 1 7R117Q-M119V-R120V-A131E-L203T-I204Y- 100% 31%  0%G206Q-S227V-E252Q-I272V R117Q-M119V-R120V-A131E-L203T-I204Y- 100% 77% 6% G206Q-S227V-R229V-E252Q-I272P R117Q-A131E-L203S-I204Y-S227V-R229Q-100% 78% 13% E252Q-I272P A131E-L203S-G206Q-A217Y-R229V-I272P 100% 100% 71% L203N-A217Y-A224S-R229A 100% 81% 22%R117Q-L203S-A217Y-A224S-S227D-R229S 100% 91% 55%R117S-L203C-G206E-A217Y-R229Q 100% 83% 34%R117Q-A131E-L203C-A217Y-S227D-R229P 100% 82% 37%A131E-G209D-A217Y-A224S-R229A 100% 82% 29%R117Q-M119V-R120V-A131E-L203T-I204Y- 100% 88% 17%G206Q-A217Y-S227D-R229V-E252Q-I272V R117G-Y127D-A131E-A205W-A217Y-V221T-100% 100%  100%  E252Q-I272K Y022F-A159D-L203R-G209D-A217Y-V221T- 100%97% 96% I272K R120Q-K121E-L203C-A217Y-R229A 100% 78% 58%A131E-L203R-A217Y-A224S-R229A 100% 100%  76%R117Q-A131E-L203R-G206E-A217Y-A224S- 100% 93% 65% S227D-R229AA131E-L203N-A217Y-A224S-S227D 100% 88% 27% R120E-G206E-A217Y-R2290 100%60% 13% R117Q-M119V-R120V-A131E-L203T-I204Y- 100% 76% 42%G206Q-A217Y-S227V-R229V-E252Q-I272P R117Q-M119V-R120V-A131E-L203T-I204Y-100% 70% 20% A205W-G206Q-S227D-R229V-E252QR117Q-M119V-R120V-A131E-L203T-I204Y- 100% 76% 48%A205W-G206Q-S227V-R229V-E252Q-I272P R117G-A131E-I204Y-A205W-G209A-A217Y-100% 73% 53% V221T-R229Q-E252Q-I272K A131E-A217Y 100% 51%  2%Y022F-R117E-A131E-L203R-G209D-A217Y 100% 72% 70%R117E-A131E-L203R-G209D-A217Y 100% 66% 57%R117E-A131E-L203R-I204Y-G209A-A217Y- 100% 63% 16% V221TY022F-R117E-A131E-F143L-A217Y-V221T 100% 76% 55%Y022F-P125E-A131E-I179K-L203R-G209D- 100% 83% 81%A217Y-V221T-R229C-I272K A131E-A217Y-V221T 100% 62% 11%A131E-L203R-G209D-A217Y-V221T 100% 100%  90%A131E-L203R-G209D-A217Y-V221T-R229C- 100% 78% 62% I272KR117Q-Y127D-A217Y-E252Q 100% 70% 45%R117E-Y127D-A131D-A217Y-V221T-E252Q- 100% 82% 76% I272PT004K-A070V-K071E-R117Q-A131T-F143L- 100%  7%  0% N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229H- N280K L203R-G209D-A217Y-R229Q 100%81% 69% R117E-Y127D-A131E-A217Y-V221T-E252Q- 100% 87% 81% I272K SEQ IDNO: 1 100%  0%  0%

That which is claimed:
 1. A lipolytic enzyme variant or an activefragment thereof comprising at least two amino acid modifications to aparent lipolytic enzyme, wherein a first amino acid modification is at aposition of the lipolytic enzyme variant selected from the groupconsisting of 4, 11, 19, 22, 26, 39, 53, 60, 64, 67, 70, 71, 93, 95,111, 117, 119, 120, 121, 125, 127, 131, 134, 141, 142, 143, 146, 149,154, 159, 163, 166, 170, 179, 185, 196, 199, 203, 204, 205, 206, 208,209, 212, 217, 218, 221, 224, 227, 229, 235, 247, 252, 261, 262, 265,272, 280 and 286, wherein the amino acid positions of the variant arenumbered by correspondence with the amino acid sequence of SEQ ID NO: 1,wherein the lipolytic enzyme variant has at least 60% sequence identityto the amino acid sequence SEQ ID NO:
 1. 2. The lipolytic enzyme variantor active fragment thereof of claim 1, wherein the at least two aminoacid modifications to a parent lipolytic enzyme are at a position of thelipolytic enzyme variant selected from the group consisting of 4, 11,19, 22, 26, 39, 53, 60, 64, 67, 70, 71, 93, 95, 111, 117, 119, 120, 121,125, 127, 131, 134, 141, 142, 143, 146, 149, 154, 159, 163, 166, 170,179, 185, 196, 199, 203, 204, 205, 206, 208, 209, 212, 217, 218, 221,224, 227, 229, 235, 247, 252, 261, 262, 265, 272, 280, and 286, whereinthe amino acid positions of the variant are numbered by correspondencewith the amino acid sequence of SEQ ID NO:
 1. 3. The lipolytic enzymevariant or an active fragment thereof of claim 1 or 2, wherein saidvariant is derived from a parent lipolytic enzyme having 60%, 65%, 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% amino acid sequence identity to the amino acidsequence of SEQ ID NO:
 1. 4. The lipolytic enzyme variant or an activefragment thereof of claim 1 or claim 2, wherein said variant comprisesan amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acidsequence identity to the amino acid sequence of SEQ ID NO:
 1. 5. Thelipolytic enzyme variant or active fragment of claim 1 or 2, wherein theamino acid sequence has at least two or more of the following featureswith respect to SEQ ID NO: 1: a K at position 4; an R at position 11; anL at position 19; an F at position 22; a C at position 26; an I atposition 39; a Q at position 53; an L at position 60; a V or T atposition 64; a G at position 67; a V at position 70; an E at position71; an S at position 93; a Q at position 95; a T at position 111; a D,E, G, Q or S at position 117; a V at position 119; an E or Q or S or Vat position 120; an E or S at position 121; an E at position 125; a D orE at position 127; a D, E, K or T at position 131; a E or M at position134; an N at position 141; a V at position 142; an L at position 143; aH at position 146; an I at position 149; a V at position 154; a D atposition 159; a G at position 163; a T at position 166; an E or S atposition 170; a K at position 179; a Q at position 185; a L at position196; an H at position 199; a C, K, N, R, S ort T at position 203; a V orY at position 204; a W at position 205; a C, E or Q at position 206; anE or Q at position 208; an A or D at position 209; a R at position 212;a Y at position 217; a D at position 218; a T at position 221; an S atposition 224; a D or V at position 227; an A, C, H, P, Q, S, T or V atposition 229; an A at position 235; an R at position 247; a D, G, Q or Vat position 252; an A or L at positon 261; a C or M at position 262; anA at position 265; a K, P or V at position 272; a K at position 280; anda Q at position 286, wherein the amino acid positions of the variant arenumbered by correspondence with the amino acid sequence of SEQ ID NO: 1.6. The lipolytic enzyme variant or an active fragment thereof of anypreceding claim, wherein the variant or active fragment thereofcomprises amino acid modifications selected from the group consisting ofY022F-L203R, A131D-L203R, Y127D-L203R, A131E-L203R, L203R-I204Y,L203R-G206C, L203R-K208Q, L203R-G209D, L203R-G209A, L203R-R229Q,L203R-E252Q, L203R-V261L, L203R-A262M, L203R-A262C, L203R-I272P,Y022F-R117D-L203R, Y022F-R117Q-L203R, Y022F-A131E-L203R,Y022F-L203R-G206C, Y022F-L203R-K208Q, Y022F-L203R-G209D,Y022F-L203R-G209A, Y022F-L203R-R229Q, Y022F-L203R-R229T,Y022F-L203R-I272P, R117D-A131E-L203R, R117D-L203R-E252Q,R117Q-A131D-L203R, A131D-L203R-G206C, A131D-L203R-R229T,A131D-L203R-E252Q, R117Q-L203R-K208Q, Y127D-L203R-K208Q,A131E-L203R-K208Q, L203R-I204Y-K208Q, L203R-K208Q-G206C,L203R-K208Q-G209D, L203R-K208Q-G209A, L203R-K208Q-R229Q,L203R-K208Q-R229T, L203R-K208Q-E252Q, L203R-K208Q-V261L,L203R-K208Q-A262M, L203R-K208Q-I272P, Y127D-L203R-G209D,A131E-L203R-G209D, L203R-I204Y-G209D, L203R-G206C-G209D,L203R-G209D-R229Q, L203R-G209D-R229T, L203R-G209D-E252Q,L203R-G209D-I272P, R117Q-L203R-R229T, Y127D-L203R-R229T,A131E-L203R-R229T, L203R-I204Y-R229T, L203R-G206C-R229T,L203R-G209A-R229T, L203R-R229T-E252Q, L203R-R229T-A262M,L203R-R229T-I272P, R117Q-L203R-I272P, Y127D-L203R-I272P,A131E-L203R-I272P, L203R-I204Y-I272P, L203R-G206C-I272P,L203R-G209A-I272P, L203R-E252Q-I272P, A131D-L203R-G209D-R229T,A131D-L203R-R229T-I272P, A131D-L203S-R229T-I272P,L203R-K208Q-G209D-R229T, L203R-K208Q-R229T-I272P,L203S-K208Q-G209D-R229T, L203S-K208Q-R229T-I272P,Y022F-R117Q-A131T-F143L-L203R-G209D-R229Q,Y199H-L203R-G209D-A217Y-R229Q, L203R-G209D-A217Y-R229Q,R117Q-L203R-G209D-R229C, Y199H-A217Y,A217Y-V221T-R229C-E252Q-A262M-I272P, E252Q-A262M-I272P,Y022F-A159D-L203R-G206C-K208Q-G209D-I272P,Y022F-L203R-G206C-K208Q-G209D, P125E-L203R-K208Q-G209D,Y022F-R229C-E252Q-I272P, A159D-L203R-G209D,T004K-K071E-R117D-A131T-F143L-N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229C-N280K,T004K-K071E-R117Q-A131D-F143L-N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229C-N280K,A159D-L203R-G209D-R229H, A159D-L203R-G209D-R229C,A159D-L203R-G209D-R229V, A159D-L203R-G206E-G209D,Y022F-R117Q-A131D-L203R-R229V-E252Q,Y022F-R053Q-R117Q-E252Q-V261L-I272P, Y022F-R117Q-A131D-E252Q,Y022F-R117Q-R229V-E252Q-V261L-I272P, Y022F-R053Q-R229V-E252Q-I272P,R117Q-A131D-E252Q-I272P, Y022F-R053Q-R117Q-R229V-V261L,Y022F-R117Q-G209D-R229V-E252Q-I272P, Y022F-A131D-R229V-E252Q-I272P,Y022F-R053Q-R117Q-L203R-R229V-E252Q-V261L, Y022F-R117Q-R229V,Y022F-R117Q-A131D-R229V-E252Q,Y022F-R053Q-R117Q-L203R-R229V-E252Q-V261L-I272P,Y022F-R053Q-R117Q-L203R-R229V-E252Q-I272P,Y022F-R053Q-R117Q-L203R-R229V-E252Q,Y022F-A131D-L203R-R229V-E252Q-I272P, Y022F-R053Q-R117Q-E252Q-I272P,Y022F-R117Q-E252Q-V261L, R117Q-A131D-L203R-R229V-E252Q,Y022F-R229V-E252Q-I272P, Y022F-R117Q-R229V-I272P, Y022F-R117D,Y022F-K134E-R229Q, Y022F-R117D-I272P, R117E-A131E-I204Y-E252Q-I272K,A131E-V221T-E252Q-I272K, R117E-A131E-I204Y-G209A-V221T-R229C,R117E-A131E-R229C, R117E-V221T-R229C,Y127D-A131E-I204Y-V221T-E252Q-I272K, Y127D-G209A-V221T-R229C,R117E-G209A-V221T-E252Q, A131E-I204Y-G209A-V221T-E252Q,R117E-Y127D-A131E-I204Y-A217Y-V221T-R229C-E252Q, R117E-A131E-I272K,R117E-A131E-G209A-V221T, Y127D-A131E-A217Y-V221T-E252Q,A131E-I204Y-V221T-I272K, Y127D-G209A-V221T-E252Q-I272K,A131E-I204Y-V221T, Y127D-A131E-I204Y-E252Q,Y127D-A131E-I204Y-G209A-V221T-I272K, Y127D-A131E-G209A-E252Q-I272K,Y127D-A131E-I204Y-G209A-V221T-R229C-E252Q,Y127D-V221T-R229C-E252Q-I272K, A131E-V221T-R229C-E252Q,Y127D-I204Y-V221T-E252Q, R117E-Y127D-A131E-A217Y-V221T-E252Q-I272K,G209A-V221T-R229C-E252Q, R117E-I204Y-V221T,Y127D-I204Y-V221T-R229C-I272K, R117E-V221T-E252Q-I272K,R117E-A131E-V221T, I204Y-V221T-E252Q-I272K,R117E-Y127D-A131E-I204Y-A217Y-V221T-E252Q-I272K,A131E-G209A-V221T-R229C-I272K,Y127D-A131E-I204Y-V221T-R229C-E252Q-I272K, R117E-V221T-I272K,Y127D-A131E-I204Y-I272K, A131E-E252Q,R117E-A131E-I204Y-G209A-V221T-E252Q-I272K,Y127D-A131E-G209A-R229C-E252Q, A131E-G209A-V221T-R229C,R117E-A131E-I204Y-G209A-V221T-I272K, R117E-A131E-I204Y-E252Q,R117E-A131E-I204Y-V221T-R229C-E252Q, A131E-G209A-V221T-I272K,Y127D-E252Q, R117E-A131E-I204Y-G209A-V221T-R229C-E252Q-I272K,R117E-V221T-R229C-E252Q, Y127D-A131E-I204Y-G209A-R229C-E252Q-I272K,R117E-A131E-I204Y-I272K, R117E-A131E-I204Y-V221T-R229C-I272K,R117E-A131E-A217Y-V221T-E252Q, I204Y-G209A-V221T-E252Q-I272K,R117E-Y127D-I204Y-E252Q, I204Y-G209A-V221T,R117E-Y127D-A131E-G209A-E252Q-I272K, R117E-A131E-R229C-E252Q,Y127D-A131E-I204Y-G209A-V221T-R229C-I272K,Y127D-A131E-I204Y-G209A-V221T-R229C,Y127D-A131E-G209A-R229C-E252Q-I272K,A131E-I204Y-G209A-V221T-E252Q-I272K, Y127D-A131E-I204Y-E252Q-I272K,Y127D-A131E-I204Y, R117E-I204Y-G209A-V221T-E252Q,R117E-A131E-G209A-V221T-R229C-E252Q, A131E-I204Y-G209A-V221T-R229C,R117E-V221T-R229C-I272K, Y127D-I204Y-G209A-E252Q-I272K,G209A-V221T-R229C-E252Q-I272K, Y127D-I204Y-V221T-R229C,Y127D-I204Y-G209A-V221T-I272K, R117E-A131E-V221T-I272K,Y127D-I204Y-G209A-V221T-E252Q, L203S-E252Q, L203S-E252Q-I272P,M119V-L203S-E252Q, L203S-S227V-E252Q, M119V-L203S-S227V-E252Q-I272P,M119V-L203S-S227V-E252Q-I272V, R117Q-M119V-L203S-S227V-E252Q-I272P,R117Q-M119V-L203S-S227V-E252Q-I272V,T142V-K185Q-L203S-S227V-E252Q-I272V-K286Q,M119V-K185Q-L203S-S227V-E252Q-I272V-K286Q,M119V-T142V-L203S-S227V-E252Q-I272V-K286Q,M119V-T142V-K185Q-S227V-E252Q-I272V-K286Q,M119V-T142V-K185Q-L203S-S227V-I272V-K286Q,M119V-T142V-K185Q-L203S-S227V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227D-R229V-E252Q,M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q-I272V,R117Q-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q-I272V,R117Q-M119V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-I204Y-G206Q-S227V-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-S227V-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227D-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q-I272P,A131E-L203K-G206Q-R229V-E252Q-I272V,A131E-L203T-G206Q-R229V-E252Q-I272V, A131E-L203K-R229V-E252Q-I272V,A131E-L203K-G206Q-E252Q-I272V, L203T-G206Q-R229V-E252Q-I272V,A131E-L203T-G206Q-R229V-I272V,S095Q-A131E-L203K-G206Q-R229V-K247R-E252Q-I272V,S095Q-L203K-G206Q-R229V-K247R-E252Q-I272V,S095Q-A131E-L203T-G206Q-R229V-K247R-E252Q-I272V,S095Q-A131E-G206Q-R229V-K247R-E252Q-I272V,S095Q-A131E-L203K-R229V-K247R-E252Q-I272V,S095Q-A131E-L203K-G206Q-K247R-E252Q-I272V,S095Q-A131E-L203K-G206Q-R229V-E252Q-I272V,S095Q-A131E-L203K-G206Q-R229V-K247R-I272V,S095Q-A131E-L203K-G206Q-R229V-K247R-E252Q,A131E-L203S-G206Q-R229V-I272P, L203S-G206Q-R229V-I272P,A131E-L203S-R229V-I272P, A131E-L203S-G206Q-I272P,A131E-L203S-G206Q-R229V, R117Q-L203T-A224S-E252Q,R117Q-A131E-L203S-I204Y-S227V-R229Q-E252Q-I272P,A131E-L203S-I204Y-S227V-R229Q-E252Q-I272P,R117Q-L203S-I204Y-S227V-R229Q-E252Q-I272P,R117Q-A131E-I204Y-S227V-R229Q-E252Q-I272P,R117Q-A131E-L203S-S227V-R229Q-E252Q-I272P,R117Q-A131E-L203S-I204Y-R229Q-E252Q-I272P,R117Q-A131E-L203S-I204Y-S227V-R229Q-I272P,R117Q-A131E-L203S-I204Y-S227V-R229Q-E252Q,A131E-L203S-G206Q-A217Y-R229V-I272P,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-A217Y-S227D-R229V-E252Q,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-A217Y-S227D-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-A217Y-S227V-R229V-E252Q-I272P,R117Q-M119V-R120V-A131E-L203T-I204Y-A205W-G206Q-S227D-R229V-E252Q,R117Q-M119V-R120V-A131E-L203T-I204Y-A205W-G206Q-S227D-R229V-E252Q-I272V,R117Q-M119V-R120V-A131E-L203T-I204Y-A205W-G206Q-S227V-R229V-E252Q-I272P,R117E-Y127D-A131E-I204Y-A205W-A217Y-V221T-R229C-E252Q,R117G-Y127D-A131E-I204Y-A217Y-V221T-R229Q-E252Q,R117G-Y127D-A131E-I204Y-A205W-A217Y-V221T-R229Q-E252Q,R117G-Y127D-A131E-A217Y-V221T-E252Q-I272K,R117G-Y127D-A131E-A205W-A217Y-V221T-E252Q-I272K,R117G-A131E-I204Y-A217Y-V221T-R229Q-E252Q,R117G-A131E-I204Y-A205W-A217Y-V221T-R229Q-E252Q,R117G-A131E-I204Y-G209A-A217Y-V221T-R229Q-E252Q-I272K,R117G-A131E-I204Y-A205W-G209A-A217Y-V221T-R229Q-E252Q-I272K,R117E-A131E-A205W-A217Y-V221T-E252Q,Y127D-A131E-I204Y-A205W-G209A-A217Y-V221T-R229Q, R117E-A131E-A217Y,A131E-A217Y, L203R-A217Y, L203R-G209D-A217Y, G209D-A217Y,Y022F-R117Q-L203R-G209D-A217Y,Y022F-A159D-L203R-G206C-K208Q-G209D-A217Y-I272P,R117E-A131E-I204Y-G209A-A217Y, R117E-A131E-I204Y-G209A-A217Y-V221T,R117E-A131E-I204Y-G209A-A217Y-V221T-R229C-E252Q-I272K,R117E-A131E-F143L-I204Y-G209A-A217Y-V221T-R229C-E252Q-I272K,Y022F-L203R-G206C-K208Q-G209D-A217Y, Y022F-R117E-A131E-L203R-G209D,Y022F-R117E-A131E-L203R-G209D-A217Y, R117E-A131E-L203R-G209D,R117E-A131E-L203R-G209D-A217Y, R117E-A131E-L203R-I204Y-G209A-V221T,R117E-A131E-L203R-I204Y-G209A-A217Y-V221T, A217Y-E252Q,R117E-A131E-I204Y-G209A-A217Y-E252Q,Y022F-R053Q-R117Q-F143L-L203R-G206C-K208Q-R229V-E252Q,Y022F-R117D-F143L-I272P, Y022F-R117E-A131E-A217Y-V221T,Y022F-R117E-A131E-F143L-A217Y-V221T, A131E-L203R-A217Y-I272P,A131E-F143L-L203R-A217Y-I272P, Y022F-R053Q-F143L-R229V-E252Q-I272P,R117E-A131E-A217Y-V221T-E252Q-I272K,R117E-Y127D-A131E-A217Y-E252Q-I272K,R117E-Y127D-A131E-A217Y-V221T-E252Q,R117E-Y127D-A131E-A217Y-V221T-I272K,R117E-Y127D-A131E-I204Y-G209A-A217Y-V221T-E252Q-I272K,R117E-Y127D-A131E-F143L-I204Y-G209A-A217Y-V221T-R229C-E252Q-I272K,Y127D-A217Y, R117E-A131E-A217Y-V221T,Y022F-A159D-L203R-G209D-A217Y-V221T-I272K,Y022F-P125E-A131E-I179K-L203R-G209D-A217Y-V221T-R229C-I272K,A131E-A217Y-V221T, A131E-G209D-V221T-I272K,A131E-L203R-G209D-A217Y-V221T,A131E-L203R-G209D-A217Y-V221T-R229C-I272K,R117E-Y127D-L203S-A217Y-E252Q, Y022F-A217Y-E252Q,Y022F-L203R-G206C-G209D-A217Y-E252Q,R117Q-A131E-L203S-G209D-A217Y-E252Q, R117Q-Y127D-A217Y-E252Q,A131D-R229V-E252Q-I272P, Y022F-A131D-A217Y-R229V-E252Q-I272P,Y022F-A131E-R229V-E252Q-I272P, Y022F-A131D-R229V-E252Q-I272K,Y022F-A131E-R229V-E252Q-I272K,R117E-Y127D-A131D-A217Y-V221T-E252Q-I272P,R117E-Y127D-A131D-A217Y-V221T-R229C-E252Q-I272P,R117E-Y127D-A131D-A217Y-V221T-R229V-E252Q-I272P,Y127D-A131E-A217Y-V221T-R229V-E252Q-I272K,Y127D-A131E-A217Y-V221T-R229C-E252Q-I272K,Y127D-A131E-A159D-A217Y-V221T-R229C-E252Q-I272K, L203R-R229V-E252Q,L203R-G209D-A217Y-E252Q, L203R-G209A-A217Y-E252Q, L203R-A217Y-R229V,L203R-G209D-A217Y-R229Q-E252Q, L203R-G209D-A217Y-R229V-E252Q,A159D-L203R-G209D-A217Y-E252Q, A159D-L203R-G209D-A217Y,A159D-L203R-G209D-A217Y-I272K, Y022F-L203R-G209D-A217Y,Y022F-L203R-G209D-A217Y-E252Q,Y127D-A131E-I204Y-A217Y-V221T-R229C-E252Q-I272K,Y127D-A131E-L203R-A217Y-V221T-R229C-E252Q-I272K,L203S-G209D-A217Y-R229A, L203N-A217Y-A224S-R229A,R117Q-L203S-A217Y-A224S-S227D-R229S, R117Q-K121S-L203R-R229C,R117Q-A131E-L203R, L203C-A217Y-A224S-R229P,R117Q-K121S-L203C-G206E-A217Y-S227D-R229S,A131E-L203R-G209D-A224S-R229P, R120Q-A131E-L203R,R117Q-L203R-A217Y-A224S-S227D-R229A, A217Y-A224S-R229A,L203R-S227D-R229Q, A131E-L203R-G209D-R229S, K121S-L203R,R117S-L203C-G206E-A217Y-R229Q, R117E-L203R-G209D-A224S-R229S,R120Q-L203C-G209D-R229S, L203R-K208E-G209D-A224S,R117S-R120Q-K121S-L203R-A217Y-A224S-R229C, L203R-A224S,R117Q-A131E-L203C-A217Y-S227D-R229P, R117S-L203S-A224S,Y127E-L203S-A217Y-R229C, R117Q-L203R-G206E-G209D-A217Y-S227D,R117S-K121S-L203R-R229P, R117S-R120Q-A131E, R117E-G209D,R117S-R120Q-Y127E-L203R-G206E-A217Y, L203R-A217Y-A224S-R229Q,R120Q-G206E-G209D-A217Y-A224S, R117E-R120Q-L203R-G209D-A217Y-A224S,A131E-G209D-A217Y-A224S-R229A, G209D-A224S,R117S-Y127E-A217Y-A224S-S227D-R229C, K121E-L203N-A217Y-R229A,Y127E-A217Y-A224S-R229S, R117S-A131E-L203S-R229S, L203R-K208E-R229A,R117Q-A131K-L203R-A224S, L203R-R229A, R120E-L203R-R229P, R120Q-A217Y,R120Q-L203N-A217Y-A224S-R229S, L203R-A217Y-A224S-R229A,L203S-K208E-G209D-A217Y-A224S-S227D-R229S, R117S-G209D-A217Y,L203S-G209D-S227D, A131E-L203R-A224S-R229A, L203R-A224S-R229P,Y127E-L203R-A217Y-R229C, R117Q-L203R-R229S, K121S-L203R-A217Y-A224S,R117S-R120Q-L203R-R229Q, R117S-R120Q-L203R, R117S-L203R-R229A,R117S-L203R-A224S, R117Q-S227D-R229A, L203C-G209D-A217Y-S227D,R120Q-L203R-A217Y, R117S-L203R-A224S-R229C, K121S-L203C-R229A,K121E-L203N-A217Y-A224S-S227D-R229P, K121E-L203R-A217Y,A131E-G209D-A217Y, L203N-A224S-S227D-R229C, K121E-L203R-R229C,R117Q-L203R-G209D-R229A, R117Q-A224S-R229C, R117S-L203R-A224S-R229A,R117Q-K121S-L203R-S227D-R229P, R117S-Y127E-L203S-A224S-S227D,R117S-L203R-R229Q, R117Q-L203R, A131E-L203R-A224S-R229S,K121S-A131E-L203R-A224S, R117S-K121S-L203C-G206E-A217Y-A224S-S227D,R117Q-G209D, R120Q-A131E-L203R-R229C,R117Q-R120Q-Y127E-L203S-A217Y-A224S, R117Q-R120Q-L203R-A217Y-A224S,R117Q-R120Q-L203R-A217Y, R120Q-L203R-A217Y-A224S-S227D-R229S,R117Q-R120E-L203R-R229Q, R117Q-R120E-L203R-A217Y-A224S,A131E-L203R-A224S, R117S-A131E-L203N-S227D, L203S-R229C,L203S-A224S-S227D-R229A, L203R-G209D-R229C, L203R-A217Y-R229A,L203R-G206E-A217Y-S227D-R229P, L203R-A217Y-R229S,R117Q-K121E-L203R-A224S-S227D,R117S-K121S-L203C-G206E-A217Y-A224S-S227D-R229S,R117S-K121S-L203N-A217Y-R229Q, R117Q-L203S-R229C,R117Q-L203R-G206E-A224S-R229S, R117Q-R120E-L203R,R120E-G206E-A217Y-R229Q, R117S-L203S-K208E,R120Q-K121E-L203C-A217Y-R229A, A131E-L203R-A217Y-A224S-R229A,R120E-L203R-G209D-R229Q, A131E-L203R-S227D-R229P,L203R-G209D-A217Y-A224S-R229S, L203N-G206E-A217Y,R120Q-L203R-G206E-A217Y-R229Q,R117Q-K121S-L203R-G206E-A217Y-S227D-R229Q, R120E-L203R-S227D-R229A,R117S-L203R-A224S-R229S, R120Q-L203R-A224S-R229C, L203R-R229S,L203R-R229P, R117Q-K121E-L203R-R229P, R117E-R120Q-L203R-A224S,R120Q-A131E-L203C-A217Y-A224S-R229S,R117Q-R120Q-L203S-A217Y-A224S-R229C,R117Q-A131E-L203R-G206E-A217Y-A224S-S227D-R229A,R120Q-K121E-L203C-A217Y-A224S, A131E-L203N-A217Y-A224S-S227D,R117E-L203R-G209D, R120Q-L203C-G206E, L203R-G209D-S227D,R117S-L203C-G206E-A217Y-A224S, R117E-K121E-L203R,R117E-A131E-L203N-G209D-A217Y, L203C-S227D, L203S-A217Y-R229Q,R117E-A217Y-S227D, R117Q-K121S-L203R-R229Q, L203R-K208E-G209D,R117S-L203S-G206E-G209D, R117S-K121E-L203R-A224S-R229P,L203C-A217Y-S227D-R229C, R117Q-K121S-L203R,R117Q-R120Q-Y127E-L203R-A217Y-A224S, K121E-L203S-A217Y-R229P,R120Q-L203R-G206E-A217Y-A224S, I154V-K170E-A217Y, V039I-A217Y,A217Y-V265A, Y026C-K208E-A217Y, E067G-I204V-A217Y, K170E-A217Y,K134E-A217Y, A217Y-V235A, F060L-A217Y-E252G, I064T-A217Y-V261A,I111T-A217Y, M166T-A217Y, A093S-R120S-A217Y, K170S-A217Y,S141N-A217Y-A218D-E252D, S141N-A217Y-E252G, I019L-N149I-F196L-A217Y,H011R-I064V-K134M-E163G-A217Y-E252V andT004K-A070V-K071E-R117Q-A131T-F143L-N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229H-N280K.7. The lipolytic enzyme variant or active fragment thereof of any ofclaims 1-6, wherein the variant or active fragment has lipolyticactivity.
 8. The lipolytic enzyme variant or active fragment thereof ofany preceding claims, wherein said lipolytic enzyme variant or activefragment thereof has an improved performance relative to the parentlipolytic enzyme, wherein the improved performance is selected from thegroup consisting of an improved wash performance, an increased detergentstability, an increased thermostability, and any one combinationthereof.
 9. The lipolytic enzyme variant or active fragment thereof ofany of claims 1-7, wherein the variant or active fragment has adetergent stability that is greater that the detergent stability of theparent lipolytic enzyme.
 10. The lipolytic enzyme variant or activefragment thereof of any of claims 1-7, wherein the variant or activefragment has a wash performance index (PI(wash)) relative to the parentlipolytic enzyme that is greater than 1.0.
 11. A composition comprisingat least one lipolytic enzyme variant or fragment according to claim 1.12. The composition according to claim 11, wherein said composition is adetergent composition.
 13. The composition according to claim 12,wherein said detergent composition is selected from a laundry detergent,a fabric softening detergent, a dishwashing detergent, and ahard-surface cleaning detergent.
 14. The composition of claim 11,wherein said composition further comprises one or more calcium ionsand/or zinc ions; one or more enzyme stabilizers; from about 0.001% toabout 1.0 weight % of said lipolytic enzyme variant(s); one or morebleaching agents; one or more adjunct materials; and/or one or moreadditional enzymes or enzyme derivatives selected from the groupconsisting of acyl transferases, alpha-amylases, beta-amylases,alpha-galactosidases, arabinosidases, aryl esterases,beta-galactosidases, carrageenases, catalases, cellobiohydrolases,cellulases, chondroitinases, cutinases, DNase or nuclease, endo-beta-1,4-glucanases, endo-beta-mannanases, esterases, exo-mannanases,galactanases, glucoamylases, hemicellulases, hyaluronidases,keratinases, laccases, lactases, ligninases, lipases, lipoxygenases,lysozymes, mannanases, oxidases, pectate lyases, pectin acetylesterases, pectinases, pentosanases, perhydrolases, peroxidases,phenoloxidases, phosphatases, phospholipases, phytases,polygalacturonases, proteases, pullulanases, reductases,rhamnogalacturonases, beta-glucanases, tannases, transglutaminases,xylan acetyl-esterases, xylanases, xyloglucanases, xylosidases,metalloproteases, nucleases, additional serine proteases, andcombinations thereof.
 15. The composition of claim 11, wherein saidcomposition is a granular, powder, solid, bar, liquid, tablet, gel,paste or unit dose composition.
 16. A DNA sequence encoding thelipolytic enzyme variant or active fragment of claim
 1. 17. Anexpression vector or cassette comprising the DNA sequence of claim 16.18. The expression vector or cassette of claim 17, wherein the DNAsequence is operably linked to a promoter.
 19. A recombinant host cellcomprising the DNA sequence of claim
 16. 20. Use of the lipolytic enzymevariant or fragment of claim 1 for hydrolyzing a lipolytic enzymesubstrate.
 21. A method for cleaning a surface comprising contacting thesurface with a composition comprising at least one lipolytic enzymevariant of claim 1, and optionally a surfactant.
 22. A method ofproducing a lipolytic enzyme variant comprising culturing the host cellcomprising a DNA sequence encoding the lipolytic enzyme variant oractive fragment of claim 1 in a culture medium under conditionsconducive for the production of the lipolytic enzyme variant andrecovering the lipolytic enzyme variant from said culture medium.
 23. Alipolytic enzyme variant or an active fragment thereof comprising atleast two amino acid modifications to a parent lipolytic enzyme, whereinthe variant or active fragment thereof comprises amino acidmodifications selected from the group consisting of L203R-G206C-I272P,P125E-L203R-K208Q-G209D, L203R-E252Q-I272P,Y022F-R117Q-A131T-F143L-L203R-G209D-R229Q, L203S-K208Q-G209D-R229T,R117Q-L203R-G209D-R229C, L203R-G209A-I272P, A131D-L203S-R229T-I272P,R117E-A131E-I204Y-E252Q-I272K, Y127D-A131E-A217Y-V221T-E252Q,L203R-I204Y-I272P, Y127D-I204Y-V221T-R229C-I272K,Y127D-A131E-I204Y-G209A-V221T-I272K,R117E-A131E-I204Y-G209A-V221T-E252Q-I272K,Y127D-V221T-R229C-E252Q-I272K, L203S-K208Q-R229T-I272P,Y127D-L203R-I272P, Y127D-I204Y-V221T-R229C,R117E-A131E-I204Y-G209A-V221T-I272K,R117E-A131E-I204Y-G209A-V221T-R229C, R117E-A131E-I204Y-I272K,A131E-L203R-K208Q, R117Q-L203R-K208Q, L203R-R229T-A262M,R117E-V221T-R229C-E252Q, A131E-G209A-V221T-R229C,G209A-V221T-R229C-E252Q, R117E-A131E-G209A-V221T-R229C-E252Q,R117E-A131E-I204Y-V221T-R229C-E252Q, R117E-I204Y-G209A-V221T-E252Q,A131E-L203R-G209D,T004K-A070V-K071E-R117Q-A131T-F143L-N146H-A159D-I179K-L203R-G206E-G209D-L212R-R229H-N280K,Y127D-A131E-I204Y-G209A-R229C-E252Q-I272K, L203R-R229T-E252Q,Y127D-A131E-I204Y-V221T-R229C-E252Q-I272K, L203R-G209D-I272P,L203R-G209D-R229Q, L203R-K208Q, L203R-K208Q-E252Q, R117Q-L203R-R229T,Y022F-R117Q-R229V-I272P, R117E-A131E-I204Y-V221T-R229C-I272K,A131D-L203R-E252Q, L203R-K208Q-R229T-I272P, R117Q-A131D-E252Q-I272P,R117E-A131E-I204Y-G209A-V221T-R229C-E252Q-I272K,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-E252Q-I272V,Y022F-R053Q-R117Q-L203R-R229V-E252Q, R117E-A131E-R229C,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-S227V-R229V-E252Q-I272P,Y127D-I204Y-G209A-E252Q-I272K, L203R-G209D, L203R-I272P,L203R-K208Q-A262M, R117E-A131E-R229C-E252Q,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-A217Y-S227D-R229V-E252Q-I272V,L203R-R229T-I272P,R117G-A131E-I204Y-A205W-G209A-A217Y-V221T-R229Q-E252Q-I272K,R117Q-A131E-L203S-I204Y-S227V-R229Q-E252Q-I272P,Y022F-R053Q-R117Q-E252Q-I272P, Y022F-L203R-R229Q,Y022F-R117Q-E252Q-V261L, L203R-G209D-E252Q, Y127D-G209A-V221T-R229C,L203R-R229Q, R117Q-L203R-I272P, L203R-K208Q-G209D-R229T,R117S-L203R-A224S-R229S, L203R-G206C-G209D, R117E-A131E-I204Y-E252Q,Y127D-A131E-I204Y-G209A-V221T-R229C, L203N-A217Y-A224S-R229A,L203R-I204Y, Y022F-L203R-R229T, Y127D-A131E-G209A-R229C-E252Q-I272K,R117Q-M119V-R120V-A131E-L203T-I204Y-G206Q-A217Y-S227V-R229V-E252Q-I272P,Y022F-R117Q-G209D-R229V-E252Q-I272P,R117Q-M119V-R120V-A131E-L203T-I204Y-A205W-G206Q-S227V-R229V-E252Q-I272P,R117S-L203C-G206E-A217Y-R229Q, Y022F-L203R-I272P andR117Q-L203S-A217Y-A224S-S227D-R229S, wherein the amino acid positions ofthe variant are numbered by correspondence with the amino acid sequenceof SEQ ID NO: 1, wherein the lipolytic enzyme variant has at least 60%sequence identity to the amino acid sequence SEQ ID NO: 1, and whereinsaid lipolytic enzyme variant or active fragment thereof has an improvedwash performance relative to the wash performance of said parentlipolytic enzyme.
 24. The lipolytic enzyme variant or active fragmentthereof of claim 23, wherein the variant or active fragment has adetergent stability that is greater that the detergent stability of theparent lipolytic enzyme set forth in SEQ ID NO:
 1. 25. The lipolyticenzyme variant or active fragment thereof of claim 23, wherein thevariant or active fragment has a wash performance index (PI(wash))relative to the parent lipolytic enzyme set forth in SEQ ID NO: 1 thatis greater than 1.0.